Engineered anti-TSLP antibody

ABSTRACT

The invention relates to binding compounds that specifically bind to human TSLP, as well as uses thereof, e.g., in the treatment of inflammatory disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/515,915,filed Sep. 17, 2009 now U.S. Pat. No. 8,232,372, which is the nationalstage filing under 35 U.S.C. §371 of International Patent ApplicationNo. PCT/US2007/025531, filed Dec. 13, 2007, which claims the benefit ofU.S. Provisional Application Ser. No. 60/869,974, filed Dec. 14, 2006;the disclosures of which are incorporated herein by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “BP06556SeqListing_ST25.txt” creation date of Mar. 3, 2010 anda size of 20.9 KB. This sequence listing submitted via EFS-Web is partof the specification and is herein incorporated by reference in itsentirely.

FIELD OF THE INVENTION

The present invention relates generally to a thymic stromallymphopoietin (TSLP) specific antibody, and uses thereof, particularlyin inflammatory, and allergic inflammatory disorders.

BACKGROUND OF THE INVENTION

The immune system functions to protect individuals from infectiveagents, e.g., bacteria, multi-cellular organisms, and viruses, as wellas from cancers. This system includes several types of lymphoid andmyeloid cells such as monocytes, macrophages, dendritic cells (DCs),eosinophils, T cells, B cells, and neutrophils. These lymphoid andmyeloid cells often produce signaling proteins known as cytokines. Theimmune response includes inflammation, i.e., the accumulation of immunecells systemically or in a particular location of the body. In responseto an infective agent or foreign substance, immune cells secretecytokines which, in turn, modulate immune cell proliferation,development, differentiation, or migration. An immune response canproduce pathological consequences, e.g., when it involves excessiveinflammation, as in allergic inflammatory disorders.

TSLP is an immune cytokine that induces dendritic cell-mediated CD4⁺ Tcell responses with a proallogenic phenotype (Gilliet et al., J. Exp.Medicine 197(8): 1059-1063 (2003). TSLP is involved in the initiation ofallergic inflammation (Watanabe et al., Nature Immunology 5: 426-434(2004); Soumelis et al., Nature Immunology 3: 673-680 (2002)).

Antibodies are being developed against a number of antigen targets thatare involved in immune diseases. The most significant limitation inusing antibodies as a therapeutic agent in vivo is the immunogenicity ofthe antibodies. As most monoclonal antibodies are derived from rodents,repeated use in humans results in the generation of an immune responseagainst the therapeutic antibody. Such an immune response results in aloss of therapeutic efficacy at a minimum and a potential fatalanaphylactic response at a maximum. Initial efforts to reduce theimmunogenicity of rodent antibodies involved the production of chimericantibodies, in which mouse variable regions were fused with humanconstant regions. Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-43. However, mice injected with hybrids of human variableregions and mouse constant regions develop a strong anti-antibodyresponse directed against the human variable region, suggesting that theretention of the entire rodent Fv region in such chimeric antibodies maystill result in unwanted immunogenicity in patients.

It is generally believed that complementarity determining region (CDR)loops of variable domains comprise the binding site of antibodymolecules. Therefore, the grafting of rodent CDR loops onto humanframeworks (i.e., humanization) was attempted to further minimize rodentsequences. Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988)Science 239:1534. However, CDR loop exchanges may not uniformly resultin an antibody with the same binding properties as the antibody oforigin. Changes in framework residues (FR), residues involved in CDRloop support, in humanized antibodies may also be required to preserveantigen binding affinity. Kabat et al. (1991) J. Immunol. 147:1709.While the use of CDR grafting and framework residue preservation in anumber of humanized antibody constructs has been reported, it isdifficult to predict if a particular sequence will result in theantibody with the desired binding, and sometimes biological, properties.See, e.g., Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029,Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4181, and Hodgson(1991) Biotechnology (NY) 9:421-5.

The present invention provides an engineered TSLP antibody and usesthereof to treat inflammatory, and particularly allergic inflammatory,disorders.

SUMMARY OF THE INVENTION

The present invention provides a binding compound that specificallybinds human and cyno TSLP, comprising: at least one antibody heavy chainvariable region, or a TSLP-binding fragment thereof, said heavy chainvariable region comprising at least one CDR sequence selected from thegroup consisting of SEQ ID NOs: 1, 2 and 3; or at least one antibodylight chain variable region, or a TSLP-binding fragment thereof, saidlight chain variable region comprising at least one CDR sequenceselected from the group consisting of SEQ ID NOs: 4, 5 and 6.

The present invention also provides a binding compound that specificallybinds to human and cyno TSLP comprising at least one antibody heavychain variable region, or a TSLP-binding fragment thereof, said heavychain variable region comprising at least one CDR sequence selected fromthe group consisting of SEQ ID NOs: 1, 2 and 3; and at least oneantibody light chain variable region, or a TSLP-binding fragmentthereof, said light chain variable region comprising at least one CDRsequence selected from the group consisting of SEQ ID NOs: 4, 5 and 6.

In some embodiments, the antibody heavy chain variable region, orTSLP-binding fragment thereof, comprises at least two CDR sequencesselected from the group consisting of SEQ ID NOs: 1, 2 and 3. In otherembodiments, the antibody heavy chain variable region, or TSLP-bindingfragment thereof, has the three CDR sequences set forth in SEQ ID NOs:1, 2 and 3.

In some embodiments, the antibody light chain variable region, orTSLP-binding fragment thereof, comprises at least two CDR sequencesselected from the group consisting of SEQ ID NOs: 4, 5 and 6. In otherembodiments, the antibody light chain variable region, or TSLP-bindingfragment thereof, has the three CDR sequences set forth in SEQ ID NOs:4, 5 and 6.

The present invention also provides a binding compound that specificallybinds human and cyno TSLP, comprising: at least one antibody heavy chainvariable region, or a TSLP-binding fragment thereof, said heavy chainvariable region comprising: the CDR-H1 of SEQ ID NO. 1, or a variantthereof; the CDR-H2 of SEQ ID NO. 2, or a variant thereof; and theCDR-H3 of SEQ ID NO. 3, or a variant thereof; or at least one antibodylight chain variable region, or a TSLP-binding fragment thereof, saidlight chain variable region comprising: the CDR-L1 of SEQ ID NO. 4, or avariant thereof; the CDR-L2 of SEQ ID NO. 5, or a variant thereof; andthe CDR-L3 of SEQ ID NO. 6, or a variant thereof. The present inventionalso provides a binding compound that specifically binds human and cynoTSLP, comprising at least one antibody heavy chain variable region, or aTSLP-binding fragment thereof, said heavy chain variable regioncomprising: the CDR-H1 of SEQ ID NO. 1, or a variant thereof; the CDR-H2of SEQ ID NO. 2, or a variant thereof; and the CDR-H3 of SEQ ID NO. 3,or a variant thereof; and at least one antibody light chain variableregion, or a TSLP-binding fragment thereof, said light chain variableregion comprising: the CDR-L1 of SEQ ID NO. 4, or a variant thereof; theCDR-L2 of SEQ ID NO. 5, or a variant thereof; and the CDR-L3 of SEQ IDNO. 6, or a variant thereof. In one embodiment, the variant comprises upto 20 conservatively modified amino acid residues. In one embodiment,the variant comprises up to 10 conservatively modified amino acidresidues. In one embodiment, the variant comprises up to 5conservatively modified amino acid residues. In one embodiment, thevariant comprises up to 3 conservatively modified amino acid residues.

In some embodiments of the above described binding compounds, all orsubstantially all of the remainder of the heavy chain variable region isall or substantially all a human Ig region; and all or substantially allof the remainder of the light chain variable region variable region isall or substantially all a human Ig region. In preferred embodiments,the remainder of the heavy chain variable region is human heavy chainamino acid sequence; and the remainder of the light chain variableregion is human light chain amino acid sequence.

The present invention also provides a binding compound that specificallybinds human and cyno TSLP, comprising: a heavy chain variable regioncomprising residues 1-116 of SEQ ID NO: 10 or a variant thereof; and alight chain variable region comprising residues 1-108 of SEQ ID NO: 12,or a variant thereof. In one embodiment, the variant comprises up to 20conservatively modified amino acid residues. In one embodiment, thevariant comprises up to 10 conservatively modified amino acid residues.In one embodiment, the variant comprises up to 5 conservatively modifiedamino acid residues. In one embodiment, the variant comprises up to 3conservatively modified amino acid residues. In one embodiment, thelight chain variable region comprises a variant wherein the amino acidat position 49 of SEQ ID NO:12 has been changed from Y to K.

The present invention also provides a binding compound that specificallybinds human and cyno TSLP, comprising: a heavy chain variable regioncomprising residues 1-116 of SEQ ID NO: 10; and a light chain variableregion comprising residues 1-108 of SEQ ID NO: 12.

The present invention also provides a binding compound that specificallybinds human and cyno TSLP, comprising: a heavy chain variable regionconsisting essentially of residues 1-116 of SEQ ID NO: 10; and a lightchain variable region consisting essentially of residues 1-108 of SEQ IDNO: 12.

The present invention also provides a binding compound that specificallybinds human and cyno TSLP, comprising: a heavy chain variable regionhaving at least 95%, 90%, 85% or 80% homology to residues 1-116 of SEQID NO: 10; and/or a light chain variable region having at least 95%,90%, 85% or 80% homology to residues 1-108 of SEQ ID NO: 12. In oneembodiment, the invention provides a binding compound that specificallybinds human and cyno TSLP, comprising: a heavy chain variable regionhaving at least 90% homology to residues 1-116 of SEQ ID NO: 10; and alight chain variable region having at least 90% homology to residues1-108 of SEQ ID NO: 12. In some embodiments, the heavy chain variableregion will comprise at least 95% homology to residues 1-116 of SEQ IDNO: 10; and the light chain variable region will comprise at least 95%homology to residues 1-108 of SEQ ID NO: 12.

In some embodiments, the binding compounds of the invention alsocomprise a heavy chain constant region and/or a light chain constantregion. In one embodiment, the heavy chain constant region comprises aγ1, γ2, γ3, or γ4 human heavy chain constant region or a variantthereof. In various embodiments the light chain constant regioncomprises a lambda or a kappa human light chain constant region.

In some embodiments, the binding compound of the invention is anantibody or an antigen binding fragment thereof. In various embodimentsthe antibody or fragment thereof of the present invention is polyclonal,monoclonal, chimeric, cyno-ized, humanized or fully human. In apreferred embodiment, the antibody is a humanized antibody or a fragmentthereof.

The present invention also contemplates that the binding fragment is anantibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH, Fv, scFv, F(ab′)₂, and a diabody. The present invention alsocontemplates that the binding compound is a nanobody, an avimer, or anaptimer.

In a preferred embodiment, the binding compound is the antibody producedby the hybridoma deposited as PTA-7951. In another embodiment, thebinding compound is not the antibody produced by the hybridoma depositedas PTA-7951.

The invention also encompasses an antibody or antigen biding fragmentthereof that specifically binds to human and cyno-TSLP comprising theheavy chain amino acid sequence of SEQ ID NO: 18, or a variant thereof;and/or a light chain amino acid sequence of SEQ ID NO:17 or a variantthereof. The invention also encompasses an antibody or antigen bidingfragment thereof that specifically binds to human and cyno-TSLPcomprising amino acids 19 to 472 of SEQ ID NO: 18, or a variant thereof;and/or amino acids 20 to 233 of SEQ ID NO:17 or a variant thereof. Inone embodiment, the variant comprises up to 20 conservatively modifiedamino acid residues. In one embodiment, the variant comprises up to 10conservatively modified amino acid residues. In one embodiment, thevariant comprises up to 5 conservatively modified amino acid residues.In one embodiment, the variant comprises up to 3 conservatively modifiedamino acid residues.

The present invention also comprises a binding compound thatspecifically binds human and cyno TSLP, wherein said binding compoundhas a KD of about 2.1 pM or less, as measured using KinExA technologyand human TSLP as the ligand. The present invention also comprises abinding compound that specifically binds human and cyno TSLP, whereinsaid binding compound has a KD of 2.1 pM (+/− two-fold), as measuredusing KinExA technology and human TSLP as the ligand. In one embodiment,the binding compound is a humanized anti-TSLP antibody or an antigenbinding fragment thereof.

The present invention also comprises a binding compound thatspecifically binds human and cyno TSLP, wherein said binding compoundhas a KD of about 111 pM or less, as measured using surface plasmonresonance and human TSLP as the ligand. The present invention alsocomprises a binding compound that specifically binds human and cynoTSLP, wherein said binding compound has a KD of 111 pM (+/− two-fold),as measured using surface plasmon resonance and human TSLP as theligand. In one embodiment, the binding compound is a humanized anti-TSLPantibody or an antigen binding fragment thereof.

The present invention also comprises a binding compound thatspecifically binds human and cyno TSLP, wherein said binding compoundhas an EC50 of about 7.6 nM or less. The present invention alsocomprises a binding compound that specifically binds human and cynoTSLP, wherein said binding compound has an EC50 of about 7.6 nM (+/−two-fold). (The EC50 refers to the concentration of binding compoundrequired to neutralize human TSPL to 50% of the level observed in theabsence of the binding compound.) In one embodiment, the bindingcompound is a humanized anti-TSLP antibody or an antigen bindingfragment thereof.

The present invention also provides an isolated nucleic acid encoding atleast one of the heavy chain variable region or light chain variableregion of the binding compound of the invention. Also provided is anexpression vector comprising the nucleic acid operably linked to controlsequences that are recognized by a host cell when the host cell istransfected with the vector. Also provided are a host cell comprisingthe expression vector.

Also provided is a method of producing a polypeptide comprising a heavychain variable region or a light chain variable region of the inventioncomprising: culturing the host cell of in culture medium underconditions wherein the nucleic acid sequence is expressed, therebyproducing polypeptides comprising the light and heavy chain variableregions; and recovering the polypeptides from the host cell or culturemedium.

The invention also provides a binding compound (for example an antibodyor antigen binding fragment thereof) that specifically binds to theepitope on human TSLP that is bound by the antibody produced by thehybridoma deposited as PTA-7951, wherein the antibody that specificallybinds to the epitope on human TSLP is not the antibody produced by thehybridoma deposited as PTA-7951.

The invention also comprises a binding compound (for example an antibodyor antigen binding fragment thereof) that competitively inhibits bindingby the antibody produced by the hybridoma deposited as PTA-7951 to humanTSLP, wherein the antibody that competitively inhibits binding is notthe antibody produced by the hybridoma deposited as PTA-7951.

The invention also comprises a binding compound (for example an antibodyor antigen binding fragment thereof) that blocks TSLP-mediated activity.TSLP mediated activities include, but are not limited to, binding to itsreceptor, promoting the activation of dendritic cells leading toproliferation or survival of T_(H)2 cells, secretion of T_(H)2attracting chemokines by dendritic cells such as TARC and MDC,production of pro-allergic cytokines such as IL-4, IL-5, IL-13 andTNF-alpha. A number of assays can be employed to determine whether abinding compound blocks TSLP-mediated activity. These include the assaysdescribed n the Examples and other assays, including those described inthe art. See, e.g., Reche et al., J. Immunol. 167:336-43 (2001); Isaksenet al., J. Immunol. 168:3288-94 (2002).

In one embodiment, the binding compound is able to block the binding ofTSLP to TSLPR in a cross-blocking assay.

The present invention encompasses a method of suppressing an immuneresponse in a human subject comprising administering to a subject inneed thereof an a binding compound that specifically binds human andcyno TSLP, in an amount effective to block the biological activity ofTSLP. The present invention also contemplates administering anadditional immunosuppressive or anti-inflammatory agent. In a preferredembodiment, the immune response is asthma. In another preferredembodiment, the immune response is allergic inflammation. In anotherpreferred embodiment, the allergic inflammation is allergicrhinosinusitis, allergic asthma, allergic conjunctivitis, or atopicdermatitis. In another preferred embodiment, the immune response isfibrosis, inflammatory bowel disease or Hodgkin's lymphoma. In anotherpreferred embodiment, the binding compound is administered incombination with another immunomodulatory agent.

The antibody or fragment thereof of the present invention can be in acomposition comprising the binding compound of the invention, incombination with a pharmaceutically acceptable carrier or diluent. In afurther embodiment, the composition further comprises animmunosuppressive or anti-inflammatory agent.

The present invention also encompasses a composition comprising abinding compound of the invention and a pharmaceutically acceptablecarrier or diluent.

The present invention encompasses an isolated nucleic acid encoding thepolypeptide sequence of the antibody or fragment thereof of the presentinvention. The nucleic acid can be in an expression vector operablylinked to control sequences recognized by a host cell transfected withthe vector. Also encompassed is a host cell comprising the vector, and amethod of producing a polypeptide comprising culturing the host cellunder conditions wherein the nucleic acid sequence is expressed, therebyproducing the polypeptide, and recovering the polypeptide from the hostcell or medium.

In various embodiments, the invention relates to medicaments comprisingthe antibody or fragment thereof of the present invention. For example,the invention encompasses the use of a binding compound thatspecifically binds human and cyno TSLP (for example, any one of thebinding compounds according to the invention) for the preparation of amedicament to treat suppress an immune response. The present inventionencompasses the use of a binding compound that specifically binds humanand cyno TSLP (for example, any one of the binding compounds accordingto the invention) for the preparation of a medicament to treat asthma.The present invention encompasses the use of a binding compound thatspecifically binds human and cyno TSLP (for example, any one of thebinding compounds according to the invention) for the preparation of amedicament to treat an inflammatory disorder. In one embodiment, theinflammatory disorder is an allergic inflammatory disorder. In oneembodiment, the allergic inflammatory disorder is allergicrhinosinusitis, allergic asthma, allergic conjunctivitis, or atopicdermatitis.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise. All referencescited herein are incorporated by reference to the same extent as if eachindividual publication, patent application, or patent, was specificallyand individually indicated to be incorporated by reference.

I. Definitions

“Activation,” “stimulation,” and “treatment,” as it applies to cells orto receptors, may have the same meaning, e.g., activation, stimulation,or treatment of a cell or receptor with a ligand, unless indicatedotherwise by the context or explicitly. “Ligand” encompasses natural andsynthetic ligands, e.g., cytokines, cytokine variants, analogues,muteins, and binding compositions derived from antibodies. “Ligand” alsoencompasses small molecules, e.g., peptide mimetics of cytokines andpeptide mimetics of antibodies. “Activation” can refer to cellactivation as regulated by internal mechanisms as well as by external orenvironmental factors. “Response,” e.g., of a cell, tissue, organ, ororganism, encompasses a change in biochemical or physiological behavior,e.g., concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Proliferative activity” encompasses an activity thatpromotes, that is necessary for, or that is specifically associatedwith, e.g., normal cell division, as well as cancer, tumors, dysplasia,cell transformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding composition, or by another cell.“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications.

As used herein, the term “antibody” refers to any form of antibody orfragment thereof that exhibits the desired biological activity. Thus, itis used in the broadest sense and specifically covers monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired biologicalactivity.

As used herein, the term “TSLP binding fragment” or “binding fragmentthereof” encompasses a fragment or a derivative of an antibody thatstill substantially retain its biological activity of inhibiting TSLPactivity. Therefore, the term “antibody fragment” or TSLP bindingfragment refers to a portion of a full length antibody, generally theantigen binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules, e.g., sc-Fv; domainantibodies; and multispecific antibodies formed from antibody fragments.Typically, a binding fragment or derivative retains at least 10% of itsTSLP inhibitory activity. Preferably, a binding fragment or derivativeretains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (ormore) of its TSLP inhibitory activity, although any binding fragmentwith sufficient affinity to exert the desired biological effect will beuseful. It is also intended that a TSLP binding fragment can includeconservative amino acid substitutions that do not substantially alterits biologic activity.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., (1975) Nature 256: 495, or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., (1991)Nature 352: 624-628 and Marks et al., (1991) J. Mol. Biol. 222: 581-597,for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, the term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

The monoclonal antibodies herein also include camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci.26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94/04678;WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby incorporated byreference in their entireties). In one embodiment, the present inventionprovides single domain antibodies comprising two V_(H) domains withmodifications such that single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The prefix “hu”or “hum” is added to antibody clone designations when necessary todistinguish humanized antibodies (e.g., “hu23B12”) from parental rodentantibodies (e.g., rat 23B12, or “r23B12”). The humanized forms of rodentantibodies will generally comprise the same CDR sequences of theparental rodent antibodies, although certain amino acid substitutionsmay be included to increase affinity or increase stability of thehumanized antibody.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies, and alonger half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” refers to an antibody which comprises mouse immunoglobulinsequences only.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variabledomain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain; Kabat et al., (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma “hypervariable loop” (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, (1987)J. Mol. Biol. 196: 901-917). As used herein, the term “framework” or“FR” residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues. Theresidue numbering above relates to the Kabat numbering system and doesnot necessarily correspond in detail to the sequence numbering in theaccompanying Sequence Listing. See Tables 2 and 3, in which sequencenumbering is with reference to the Sequence Listing.

“Binding” refers to an association of the binding composition with atarget where the association results in reduction in the normal Brownianmotion of the binding composition, in cases where the bindingcomposition can be dissolved or suspended in solution.

“Binding compound” refers to a molecule that comprises one or more aminoacid sequences that specifically bind to human TSLP. In one preferredembodiment, the binding compound is an antibody. In another preferredembodiment, the binding compound comprises an antibody fragment.

“Binding composition” refers to a TSLP-binding compound in combinationwith a stabilizer, excipient, salt, buffer, solvent, or additive,capable of binding to a target.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids are known to those of skill in this artand may be made generally without altering the biological activity ofthe resulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson, et al., Molecular Biology of the Gene, The Benjamin/CummingsPub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions arepreferably made in accordance with those set forth in Table 1 asfollows:

TABLE 1 Exemplary Conservative Amino Acid Substitutions OriginalConservative residue substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn(N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp;Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys(K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser(S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

The terms “consists essentially of,” or variations such as “consistessentially of” or “consisting essentially of,” as used throughout thespecification and claims, indicate the inclusion of any recited elementsor group of elements, and the optional inclusion of other elements, ofsimilar or different nature than the recited elements, that do notmaterially change the basic or novel properties of the specified dosageregimen, method, or composition. As a nonlimiting example, an antibodyor fragment thereof that consists essentially of a recited amino acidsequence may also include one or more amino acids, includingsubstitutions of one or more amino acid residues, that do not materiallyaffect the properties of the binding compound.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Effective amountalso means an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular patient or veterinary subject may varydepending on factors such as the condition being treated, the overallhealth of the patient, the method route and dose of administration andthe severity of side affects (see, e.g., U.S. Pat. No. 5,888,530 issuedto Netti, et al.). An effective amount can be the maximal dose or dosingprotocol that avoids significant side effects or toxic effects. Theeffect will result in an improvement of a diagnostic measure orparameter by at least 5%, usually by at least 10%, more usually at least20%, most usually at least 30%, preferably at least 40%, more preferablyat least 50%, most preferably at least 60%, ideally at least 70%, moreideally at least 80%, and most ideally at least 90%, where 100% isdefined as the diagnostic parameter shown by a normal subject (see,e.g., Maynard, et al. (1996) A Handbook of SOPs for Good ClinicalPractice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context.

“Endogenous” refers to substances that are produced within a cell,organism, or human body, depending on the context.

As used herein, the term “isolated nucleic acid molecule” refers to anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the antibody nucleic acid. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are distinguishedfrom the nucleic acid molecule as it exists in natural cells. However,an isolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al. (1987) Cold Spring HarborSymp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (StocktonPress, N.Y.) As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

As used herein, the term “germline sequence” refers to a sequence ofunrearranged immunoglobulin DNA sequences. Any suitable source ofunrearranged immunoglobulin DNA may be used.

“Inhibitors” are compounds that decrease, block, prevent, delayactivation, inactivate, desensitize, or down regulate, e.g., a gene,protein, ligand, receptor, or cell. An inhibitor may also be defined asa composition that reduces, blocks, or inactivates a constitutiveactivity. An “antagonist” is a compound that opposes the actions of anagonist. An antagonist prevents, reduces, inhibits, or neutralizes theactivity of an agonist. An antagonist can also prevent, inhibit, orreduce constitutive activity of a target, e.g., a target receptor, evenwhere there is no identified agonist.

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activating or inhibiting agent and are compared tocontrol samples without the agent. Control samples, i.e., not treatedwith agent, are assigned a relative activity value of 100%. Inhibitionis achieved when the activity value relative to the control is about 90%or less, typically 85% or less, more typically 80% or less, mosttypically 75% or less, generally 70% or less, more generally 65% orless, most generally 60% or less, typically 55% or less, usually 50% orless, more usually 45% or less, most usually 40% or less, preferably 35%or less, more preferably 30% or less, still more preferably 25% or less,and most preferably less than 25%.

Endpoints in inhibition can be monitored as follows. Inhibition, andresponse to treatment, e.g., of a cell, physiological fluid, tissue,organ, and animal or human subject, can be monitored by an endpoint. Theendpoint may comprise a predetermined quantity or percentage of, e.g.,an indicia of inflammation, oncogenicity, or cell degranulation orsecretion, such as the release of a cytokine, toxic oxygen, or aprotease. The endpoint may comprise, e.g., a predetermined quantity ofion flux or transport; cell migration; cell adhesion; cellproliferation; potential for metastasis; cell differentiation; andchange in phenotype, e.g., change in expression of gene relating toinflammation, apoptosis, transformation, cell cycle, or metastasis (see,e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158; Hood and Cheresh(2002) Nature Rev. Cancer 2:91-100; Timme, et al. (2003) Curr. DrugTargets 4:251-261; Robbins and Itzkowitz (2002) Med. Clin. North Am.86:1467-1495; Grady and Markowitz (2002) Annu. Rev. Genomics Hum. Genet.3:101-128; Bauer, et al. (2001) Glia 36:235-243; Stanimirovic and Satoh(2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the protein,e.g., TSLP, in a heterogeneous population of proteins and/or otherbiologics. Thus, under designated conditions, a specified ligand/antigenbinds to a particular receptor/antibody and does not bind in asignificant amount to other proteins present in the sample.

The antibody, or binding composition derived from the antigen-bindingsite of an antibody, of the contemplated method binds to its antigenwith an affinity that is at least two fold greater, preferably at leastten times greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with unrelatedantigens. In a preferred embodiment the antibody will have an affinitythat is greater than about 10⁹ liters/mol, as determined, e.g., byScatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239).

As used herein, the term “inflammatory disorder” refers to any diseaseor disorder characterized by local inflammation at a site of injury orinfection and includes, without limitation, allergic inflammation,autoimmune diseases, and other disorders characterized by undesiredimmune cell accumulation at a local tissue site.

As used herein, the term “immunomodulatory agent” refers to natural orsynthetic agents that suppress or modulate an immune response. Theimmune response can be a humoral or cellular response. Immunomodulatoryagents encompass immunosuppressive or anti-inflammatory agents.

“Immunosuppressive agents,” “immunosuppressive drugs,” or“immunosuppressants” as used herein are therapeutics that are used inimmunosuppressive therapy to inhibit or prevent activity of the immunesystem. Clinically they are used to prevent the rejection oftransplanted organs and tissues (e.g. bone marrow, heart, kidney,liver), and/or in the treatment of autoimmune diseases or diseases thatare most likely of autoimmune origin (e.g. rheumatoid arthritis,myasthenia gravis, systemic lupus erythematosus, ulcerative colitis,multiple sclerosis). Immunosuppressive drugs can be classified into fourgroups: glucocorticoids cytostatics; antibodies (including BiologicalResponse Modifiers or DMARDs); drugs acting on immunophilins; otherdrugs, including known chemotherpeutic agents used in the treatment ofproliferative disorders. For multiple sclerosis, in particular, theantibodies of the present invention can be administered in conjunctionwith a new class of myelin binding protein-like therapeutics, known ascopaxones.

“Anti-inflammatory agents” or “anti-inflammatory drugs”, is used torepresent both steroidal and non-steroidal therapeutics. Steroids, alsoknown as corticosteroids, are drugs that closely resemble cortisol, ahormone produced naturally by adrenal glands. Steroids are used as themain treatment for certain inflammatory conditions, such as: Systemicvasculitis (inflammation of blood vessels); and Myositis (inflammationof muscle). Steroids might also be used selectively to treatinflammatory conditions such as: rheumatoid arthritis (chronicinflammatory arthritis occurring in joints on both sides of the body);systemic lupus erythematosus (a generalized disease caused by abnormalimmune system function); Sjögren's syndrome (chronic disorder thatcauses dry eyes and a dry mouth).

Non-steroidal anti-inflammatory drugs, usually abbreviated to NSAIDs,are drugs with analgesic, antipyretic and anti-inflammatory effects—theyreduce pain, fever and inflammation. The term “non-steroidal” is used todistinguish these drugs from steroids, which (amongst a broad range ofother effects) have a similar eicosanoid-depressing, anti-inflammatoryaction. NSAIDs are generally indicated for the symptomatic relief of thefollowing conditions: rheumatoid arthritis; osteoarthritis; inflammatoryarthropathies (e.g. ankylosing spondylitis, psoriatic arthritis,Reiter's syndrome); acute gout; dysmenorrhoea; metastatic bone pain;headache and migraine; postoperative pain; mild-to-moderate pain due toinflammation and tissue injury; pyrexia; and renal colic. NSAIDs includesalicylates, arlyalknoic acids, 2-arylpropionic acids (profens),N-arylanthranilic acids (fenamic acids), oxicams, coxibs, andsulphonanilides.

II. General

The present invention provides engineered anti-TSLP antibodies and usesthereof to treat inflammatory, and particularly allergic inflammatory,disorders. In a preferred embodiment, the inflammatory disorder isasthma. In a preferred embodiment, the allergic inflammatory disorder isallergic rhinosinusitis, allergic asthma, allergic conjunctivitis, oratopic dermatitis. The present invention also provides engineeredanti-TSLP antibodies to treat fibrosis, inflammatory bowel disease orHodgkin's lymphoma.

TSLP is a member of the ‘long chain’ family of hematopoetic cytokines.Insights into the structural basis of ‘long chain’ cytokine/receptorrecognition have shown that although large areas of protein surface areburied in formation of cytokine—receptor complexes, the affinity of theinteraction is dominated by a few, often tightly clustered amino acidresidues forming an energetic ‘hot spot’ in the center of the bindinginterface. The identity of the residues that dominate the binding energyof a large protein-protein interface has been termed the ‘functionalepitope’. The affinity of the interaction (and hence biologicalspecificity) is consequently defined by the structural complementarityof the functional epitopes of ligand and receptor. Detailed mutagenesisstudies have shown that the most significant residues that make up thefunctional epitopes of cytokines and receptors are hydrophobic contactsinvolving either non-polar side chains such as tryptophan, the aliphaticcomponents of non-polar side chains or the polypeptide backbone. Thenon-polar ‘core’ is surrounded by a halo of polar residues of lesserimportance for binding energy. Kinetic studies indicate that the primaryrole of the functional epitopes is to stabilize protein-proteininteraction by decreasing the dissociation rate of the complex. It hasbeen suggested that the initial contact between cytokine and receptor isdominated by random diffusion or ‘rolling’ of protein surfaces producingmany unstable contacts. The complex is then stabilized when thefunctional epitopes of the receptor and ligand engage (see, e.g., Bravoand Heath, supra).

III. Generation of TSLP Specific Antibodies

Any suitable method for generating monoclonal antibodies may be used.For example, a recipient may be immunized with a linked or unlinked(e.g. naturally occurring) form of the TSLP heterodimer, or a fragmentthereof. Any suitable method of immunization can be used. Such methodscan include adjuvants, other immunostimulants, repeated boosterimmunizations, and the use of one or more immunization routes.

Any suitable source of TSLP can be used as the immunogen for thegeneration of the non-human antibody, of the compositions and methodsdisclosed herein. Such forms include, but are not limited to wholeprotein, including linked and naturally occurring heterodimers,peptide(s), and epitopes, generated through recombinant, synthetic,chemical or enzymatic degradation means known in the art.

Any form of the antigen can be used to generate the antibody that issufficient to generate a biologically active antibody. Thus, theeliciting antigen may be a single epitope, multiple epitopes, or theentire protein alone or in combination with one or more immunogenicityenhancing agents known in the art. The eliciting antigen may be anisolated full-length protein, a cell surface protein (e.g., immunizingwith cells transfected with at least a portion of the antigen), or asoluble protein (e.g., immunizing with only the extracellular domainportion of the protein). The antigen may be produced in a geneticallymodified cell. The DNA encoding the antigen may genomic or non-genomic(e.g., cDNA) and encodes at least a portion of the extracellular domain.As used herein, the term “portion” refers to the minimal number of aminoacids or nucleic acids, as appropriate, to constitute an immunogenicepitope of the antigen of interest. Any genetic vectors suitable fortransformation of the cells of interest may be employed, including butnot limited to adenoviral vectors, plasmids, and non-viral vectors, suchas cationic lipids.

Any suitable method can be used to elicit an antibody with the desiredbiologic properties to inhibit TSLP. It is desirable to preparemonoclonal antibodies (mAbs) from various mammalian hosts, such as mice,rodents, primates, humans, etc. Description of techniques for preparingsuch monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane (1988)ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York,N.Y. Thus, monoclonal antibodies may be obtained by a variety oftechniques familiar to researchers skilled in the art. Typically, spleencells from an animal immunized with a desired antigen are immortalized,commonly by fusion with a myeloma cell. See Kohler and Milstein (1976)Eur. J. Immunol. 6:511-519. Alternative methods of immortalizationinclude transformation with Epstein Barr Virus, oncogenes, orretroviruses, or other methods known in the art. See, e.g., Doyle, etal. (eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE:LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y. Coloniesarising from single immortalized cells are screened for production ofantibodies of the desired specificity and affinity for the antigen, andyield of the monoclonal antibodies produced by such cells may beenhanced by various techniques, including injection into the peritonealcavity of a vertebrate host. Alternatively, one may isolate DNAsequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according, e.g.,to the general protocol outlined by Huse, et al. (1989) Science246:1275-1281.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse et al., Science246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989). Thepolypeptides and antibodies of the present invention may be used with orwithout modification, including chimeric or humanized antibodies.Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties,magnetic particles, and the like. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinantimmunoglobulins may be produced, see Cabilly U.S. Pat. No. 4,816,567;and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; ormade in transgenic mice, see Mendez et al. (1997) Nature Genetics15:146-156; also see Abgenix and Medarex technologies.

Antibodies or binding compositions against predetermined fragments ofTSLP can be raised by immunization of animals with conjugates of thepolypeptide, fragments, peptides, or epitopes with carrier proteins.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to normal ordefective TSLP. These monoclonal antibodies will usually bind with atleast a K_(d) of about 1 μM, more usually at least about 300 nM,typically at least about 30 nM, preferably at least about 10 nM, morepreferably at least about 3 nM or better, usually determined by ELISA.

IV. Humanization of TSLP Specific Antibodies

Any suitable non-human antibody can be used as a source for thehypervariable region. Sources for non-human antibodies include, but arenot limited to, murine, Lagomorphs (including rabbits), bovine, andprimates. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance of thedesired biological activity. For further details, see Jones et al.(1986) Nature 321:522-525; Reichmann et al. (1988) Nature 332:323-329;and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

Methods for recombinantly engineering antibodies have been described,e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S.Pat. No. 4,816,567), Law et al. (European Patent Application PublicationNo. 438 310) and Winter (European Patent Application Publication No.239400).

Amino acid sequence variants of humanized anti-TSLP antibody areprepared by introducing appropriate nucleotide changes into thehumanized anti-TSLP antibody DNA, or by peptide synthesis. Such variantsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences shown for thehumanized anti-TSLP F(ab). Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of the humanizedanti-TSLP antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of thehumanized anti-TSLP antibody polypeptide that are preferred locationsfor mutagenesis is called “alanine scanning mutagenesis,” as describedby Cunningham and Wells (1989) Science 244: 1081-1085. Here, a residueor group of target residues are identified (e.g., charged residues suchas Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with TSLP antigen. The amino acidresidues demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, Ala scanning or random mutagenesis isconducted at the target codon or region and the expressed humanizedanti-TSLP antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includehumanized anti-TSLP antibody with an N-terminal methionyl residue or theantibody fused to an epitope tag. Other insertional variants of thehumanized anti-TSLP antibody molecule include the fusion to the N- orC-terminus of humanized anti-TSLP antibody of an enzyme or a polypeptidewhich increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the humanized anti-TSLPantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable loops, but FR alterations are also contemplated.Hypervariable region residues or FR residues involved in antigen bindingare generally substituted in a relatively conservative manner.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Yet another type of amino acid variant is the substitution of residuesto provide for greater chemical stability of the final humanizedantibody. For example, an asparagine (N) residue may be changed toreduce the potential for formation of isoaspartate at any NG sequenceswithin a rodent CDR. In one embodiment, the asparagine is changed toglutamine (Q). Isoaspartate formation may debilitate or completelyabrogate binding of an antibody to its target antigen. Presta (2005) J.Allergy Clin. Immunol. 116:731 at 734. In addition, methionine residuesin rodent CDRs may be changed to reduce the possibility that themethionine sulfur would oxidize, which could reduce antigen bindingaffinity and also contribute to molecular heterogeneity in the finalantibody preparation. Id. In one embodiment, the methionine is changedto alanine (A). Antibodies with such substitutions are subsequentlyscreened to ensure that the substitutions do not decrease TSLP bindingaffinity to unacceptable levels.

Nucleic acid molecules encoding amino acid sequence variants ofhumanized TSLP specific antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofhumanized anti-TSLP antibody.

Ordinarily, amino acid sequence variants of the humanized anti-TSLPantibody will have an amino acid sequence having at least 75% amino acidsequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,and most preferably at least 95%. Identity or homology with respect tothis sequence is defined herein as the percentage of amino acid residuesin the candidate sequence that are identical with the humanizedanti-TSLP residues, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, theantibody is an IgG antibody. Any isotype of IgG can be used, includingIgG₁, IgG₂, IgG₃, and IgG₄. Variants of the IgG isotypes are alsocontemplated. The humanized antibody may comprise sequences from morethan one class or isotype. Optimization of the necessary constant domainsequences to generate the desired biologic activity is readily achievedby screening the antibodies in the biological assays described below.

Likewise, either class of light chain can be used in the compositionsand methods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compositions and methods.

Any suitable portion of the CDR sequences from the non-human antibodycan be used. The CDR sequences can be mutagenized by substitution,insertion or deletion of at least one residue such that the CDR sequenceis distinct from the human and non-human antibody sequence employed. Itis contemplated that such mutations would be minimal. Typically, atleast 75% of the humanized antibody residues will correspond to those ofthe non-human CDR residues, more often 90%, and most preferably greaterthan 95%.

Any suitable portion of the FR sequences from the human antibody can beused. The FR sequences can be mutagenized by substitution, insertion ordeletion of at least one residue such that the FR sequence is distinctfrom the human and non-human antibody sequence employed. It iscontemplated that such mutations would be minimal. Typically, at least75% of the humanized antibody residues will correspond to those of thehuman FR residues, more often 90%, and most preferably greater than 95%.

CDR and FR residues are determined according to the standard sequencedefinition of Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda Md.(1987). Table 2 provides sequence identifier information for the rat(r23B12) and human (hu23B12) variable heavy chain CDRs. Table 3 providessequence identifier information for the r23B12 and hu23B12 variablelight chain CDRs.

TABLE 2 Heavy Chain Sequences and Domains ANTIBODY SEQ ID V_(H) HEAVYCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-H1 CDR-H2 CDR-H3 r23B12 71-116 26-35 50-65 95-105 hu23B12 10 1-116 26-35 50-65 95-105

TABLE 3 Light Chain Sequences and Domains ANTIBODY SEQ ID V_(L) LIGHTCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-L1 CDR-L2 CDR-L3 r23B12 81-108 24-34 50-56 89-97 hu23B12 12 1-108 24-34 50-56 89-97

The r23B12 and hu23B12 CDR-H1 sequence is GYIFTDYAMH (SEQ ID NO. 1). Ther23B12 and hu23B12 CDR-H2 sequence is TFIPLLDTSDYNQNFKG (SEQ ID NO. 2).The r23B12 and hu23B12 CDR-H3 sequence is MGVTHSYVMDA (SEQ ID NO. 3).

The r23B12 and hu23B12 CDR-L1 sequence is RASQPISISVH (SEQ ID NO. 4).The r23B12 and hu23B12 CDR-L2 sequence is FASQSIS (SEQ ID NO. 5). Ther23B12 and hu23B12 CDR-L3 sequence is QQTFSLPYT (SEQ ID NO. 6).

The r23B12 variable heavy chain amino acid sequence is EEKLQQSGDD LVRPGAAVKMSCKASGYIFTDYAMHWVKQRPGQGLEWIGTFIPLLDTSDYNQNFKGRATLTADKSSNTAYMELSRLTSEDSAVYYCARMGVTHSYVMDAWGQGASVTVS S (SEQ ID NO.7).

The r23B12 variable light chain amino acid sequence is DIVLTQSPATLSVTPGESVSLSCRASQPISISVHWFQQKSNESPRLLIKFASQSISGIPSRFSGSGSGTDFTLNINRVESEDFSVYYCQQTFSLPYTFGTGTKLELKR (SEQ ID NO. 8).

The nucleic acid sequence for the hu23B12 variable heavy chain is CAGGTG CAG CTG GTG CAG TCT GGC GCT GAG GTG AAG AAG CCT GGC GCC TCC GTG AAGGTC TCC TGC AAG GCT TCT GGC TAC ATC TTC ACC GAC TAC GCC ATG CAC TGG GTGCGG CAG GCC CCT GGC CAG GGG CTG GAG TGG ATG GGT ACC TTC ATC CCT CTG CTGGAC ACC AGC GAC TAC AAC CAG AAC TTC AAG GGC AGA GTC ACC ATG ACC ACA GACACA TCC ACC AGC ACA GCC TAC ATG GAG CTG AGG AGC CTG AGA TCT GAC GAC ACCGCC GTG TAT TAC TGT GCC AGA ATG GGA GTG ACC CAC AGC TAC GTG ATG GAT GCATGG GGC CAG GGC ACC CTG GTC ACC GTC TCC AGC (SEQ ID NO: 9), whichencodes the hu23B12 variable heavy chain amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYIFTDYAMHWVRQAPGQGLEWMGTFIPLLDTSDYNQNFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARMGVTHSYVM DAWGQGTLVTVSS (SEQ IDNO. 10).

The nucleic acid sequence for the hu23B12 variable light chain is GAAATT GTG CTG ACT CAG AGC CCA GGC ACC CTG TCT CTG TCT CCA GGC GAG AGA GCCACC CTC TCC TGC CGG GCC AGC CAG CCC ATC TCC ATC AGC GTG CAC TGG TAC CAGCAG AAA CCA GGA CAG GCT CCA AGG CTG CTG ATC TAC TTT GCC TCC CAG AGC ATCTCC GGG ATC CCC GAT AGG TTC AGC GGA TCC GGA TCT GGG ACA GAT TTC ACC CTCACC ATC AGC AGA CTG GAG CCT GAA GAT TTC GCA GTG TAT TAC TGT CAG CAG ACCTTC AGC CTG CCT TAC ACT TTC GGC CAA GGG ACC AAG GTG GAG ATC AAG CGT (SEQID NO: 11), which encodes the hu23B12 variable light chain amino acidsequence EIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGQAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTFSLPYTFGQG TKVEIKR (SEQID NO. 12).

The nucleic acid sequence for the hu23B12 heavy chain is CAG GTG CAG CTGGTG CAG TCT GGC GCT GAG GTG AAG AAG CCT GGC GCC TCC GTG AAG GTC TCC TGCAAG GCT TCT GGC TAC ATC TTC ACC GAC TAC GCC ATG CAC TGG GTG CGG CAG GCCCCT GGC CAG GGG CTG GAG TGG ATG GGT ACC TTC ATC CCT CTG CTG GAC ACC AGCGAC TAC AAC CAG AAC TTC AAG GGC AGA GTC ACC ATG ACC ACA GAC ACA TCC ACCAGC ACA GCC TAC ATG GAG CTG AGG AGC CTG AGA TCT GAC GAC ACC GCC GTG TATTAC TGT GCC AGA ATG GGA GTG ACC CAC AGC TAC GTG ATG GAT GCA TGG GGC CAGGGC ACC CTG GTC ACC GTC TCC AGC GCT AGC ACC AAG GGC CCA TCG GTC TTC CCCCTG GCA CCC TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTGGTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTGACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC TCCCTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATCTGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCCAAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA CTC CTGGGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATCTCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CCTGAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACAAAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACCGTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AACAAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCCCGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG AACCAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTGGAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTGCTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AGCAGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CACAAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA (SEQ ID NO: 13),which encodes the hu23B12 heavy chain amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYIFTDYAMHWVRQAPGQGLEWMGTFIPLLDTSDYNQNFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARMGVTHSYVMDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14).

The nucleic acid sequence for the hu23B12 light chain is GAA ATT GTG CTGACT CAG AGC CCA GGC ACC CTG TCT CTG TCT CCA GGC GAG AGA GCC ACC CTC TCCTGC CGG GCC AGC CAG CCC ATC TCC ATC AGC GTG CAC TGG TAC CAG CAG AAA CCAGGA CAG GCT CCA AGG CTG CTG ATC TAC TTT GCC TCC CAG AGC ATC TCC GGG ATCCCC GAT AGG TTC AGC GGA TCC GGA TCT GGG ACA GAT TTC ACC CTC ACC ATC AGCAGA CTG GAG CCT GAA GAT TTC GCA GTG TAT TAC TGT CAG CAG ACC TTC AGC CTGCCT TAC ACT TTC GGC CAA GGG ACC AAG GTG GAG ATC AAG CGT ACG GTG GCT GCACCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA ACT GCCTCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGGAAG GTG GAT AAC GCC CTC CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAGGAC AGC AAG GAC AGC ACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCAGAC TAC GAG AAA CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGCTCG CCC GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT (SEQ ID NO: 15), whichencodes the hu23B12 light chain amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGQAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTFSLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16).

Also contemplated are chimeric antibodies. As noted above, typicalchimeric antibodies comprise a portion of the heavy and/or light chainidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

Bispecific antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan, et al. (1985) Science 229: 81. Bispecific antibodies includebispecific antibody fragments. See, e.g., Hollinger, et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90: 6444-48, Gruber, et al., J. Immunol. 152:5368 (1994).

In yet other embodiments, different constant domains may be appended tothe humanized V_(L) and V_(H) regions provided herein. For example, if aparticular intended use of an antibody (or fragment) of the presentinvention were to call for altered effector functions, a heavy chainconstant domain other than IgG1 may be used. Although IgG1 antibodiesprovide for long half-life and for effector functions, such ascomplement activation and antibody-dependent cellular cytotoxicity, suchactivities may not be desirable for all uses of the antibody. In suchinstances an IgG4 constant domain, for example, may be used.

V. Biological Activity of Humanized Anti-TSLP

Antibodies having the characteristics identified herein as beingdesirable in a humanized anti-TSLP antibody can be screened forinhibitory biologic activity in vitro or for suitable binding affinity.To screen for antibodies that bind to the epitope on human TSLP bound byan antibody of interest (e.g., those which block binding of the cytokineto its receptor), a routine cross-blocking assay such as that describedin ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. Antibodies that bind tothe same epitope are likely to cross-block in such assays, but not allcross-blocking antibodies will necessarily bind at precisely the sameepitope since cross-blocking may result from steric hindrance ofantibody binding by antibodies bound at nearby, or even overlapping,epitopes.

Alternatively, epitope mapping, e.g., as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. “Alanine scanningmutagenesis,” as described by Cunningham and Wells (1989) Science 244:1081-1085, or some other form of point mutagenesis of amino acidresidues in human TSLP may also be used to determine the functionalepitope for an anti-TSLP antibody of the present invention. Mutagenesisstudies, however, may also reveal amino acid residues that are crucialto the overall three-dimensional structure of TSLP but that are notdirectly involved in antibody-antigen contacts, and thus other methodsmay be necessary to confirm a functional epitope determined using thismethod.

The epitope bound by a specific antibody may also be determined byassessing binding of the antibody to peptides comprising fragments ofhuman TSLP. The amino acid sequence of human TSLP is set forth in SEQ IDNO: 4 in WO 00/17362. A series of overlapping peptides encompassing thesequence of TSLP may be synthesized and screened for binding, e.g. in adirect ELISA, a competitive ELISA (where the peptide is assessed for itsability to prevent binding of an antibody to TSLP bound to a well of amicrotiter plate), or on a chip. Such peptide screening methods may notbe capable of detecting some discontinuous functional epitopes, i.e.functional epitopes that involve amino acid residues that are notcontiguous along the primary sequence of the TSLP polypeptide chain.

The epitope bound by antibodies of the present invention may also bedetermined by structural methods, such as X-ray crystal structuredetermination (e.g., WO2005/044853), molecular modeling and nuclearmagnetic resonance (NMR) spectroscopy, including NMR determination ofthe H-D exchange rates of labile amide hydrogens in TSLP when free andwhen bound in a complex with an antibody of interest (Zinn-Justin et al.(1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993)Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody: antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Additional antibodies binding to the same epitope as an antibody of thepresent invention may be obtained, for example, by screening ofantibodies raised against TSLP for binding to the epitope, or byimmunization of an animal with a peptide comprising a fragment of humanTSLP comprising the epitope sequence. Antibodies that bind to the samefunctional epitope might be expected to exhibit similar biologicalactivities, such as blocking receptor binding, and such activities canbe confirmed by functional assays of the antibodies.

Antibody affinities (e.g. for human TSLP) may be determined usingstandard analysis. Preferred humanized antibodies are those which bindhuman TSLP with a K_(D) value of no more than about 1×10⁻⁷; preferablyno more than about 1×10⁻⁸; more preferably no more than about 1×10⁻⁹;and most preferably no more than about 1×10⁻¹° M.

The antibodies and fragments thereof useful in the present compositionsand methods are biologically active antibodies and fragments. As usedherein, the term “biologically active” refers to an antibody or antibodyfragment that is capable of binding the desired the antigenic epitopeand directly or indirectly exerting a biologic effect. Typically, theseeffects result from the failure of TSLP to bind its receptor. In oneembodiment, the antibody and fragments thereof useful in the presentcompositions and methods inhibit: hTSLP induced proliferation of a Baf-3cell line transfected with hTSLP-receptor and IL-7Ralpha; hTSLP inducedluciferase expression from a Baf-3 cell line transfected with theTSLP-receptor and a luciferase reporter system; hTSLP induced TARCsecretion from human primary monocytes isolated from PBMCs; andinduction of Th2 differentiation.

As used herein, the term “specific” refers to the selective binding ofthe antibody to the target antigen epitope. Antibodies can be tested forspecificity of binding by comparing binding to TSLP to binding toirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to TSLP at least 10, and preferably 50 times morethan to irrelevant antigen or antigen mixture then it is considered tobe specific. An antibody that “specifically binds” to TSLP does not bindto proteins that do not comprise the TSLP-derived sequences, i.e.“specificity” as used herein relates to TSLP specificity, and not anyother sequences that may be present in the protein in question. Forexample, as used herein, an antibody that “specifically binds” to TSLPwill typically bind to FLAG-h TSLP, which is a fusion protein comprisingTSLP and a FLAG® peptide tag, but it does not bind to the FLAG® peptidetag alone or when it is fused to a protein other than TSLP.

VI. Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions, the antibody orfragment thereof is admixed with a pharmaceutically acceptable carrieror excipient, see, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.(1984). Formulations of therapeutic and diagnostic agents may beprepared by mixing with physiologically acceptable carriers, excipients,or stabilizers in the form of, e.g., lyophilized powders, slurries,aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001)Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with an immunosuppressive agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio between LD₅₀ and ED₅₀. Antibodies exhibiting high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a range of dosage for usein humans. The dosage of such compounds lies preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

Suitable routes of administration include parenteral administration,such as intramuscular, intravenous, or subcutaneous administration.Administration of antibody used in the pharmaceutical composition or topractice the method of the present invention can be carried out in avariety of conventional ways, such as oral ingestion, inhalation,topical application or cutaneous, subcutaneous, intraperitoneal,parenteral, intraarterial or intravenous injection. In one embodiment,the binding compound of the invention is administered intravenously. Inanother embodiment, the binding compound of the invention isadministered subcutaneously.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available (see, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003)New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, etal. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000) New Engl.J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing an inflammatory, autoimmune, orproliferative response to the reagent.

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses may be provided intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A preferred doseprotocol is one involving the maximal dose or dose frequency that avoidssignificant undesirable side effects. A total weekly dose is generallyat least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, mostgenerally at least 0.5 μg/kg, typically at least 1 μg/kg, more typicallyat least 10 μg/kg, most typically at least 100 μg/kg, preferably atleast 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably atleast 2.0 mg/kg, optimally at least 10 mg/kg, more optimally at least 25mg/kg, and most optimally at least 50 mg/kg (see, e.g., Yang, et al.(2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J.Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother.52:133-144). The desired dose of a small molecule therapeutic, e.g., apeptide mimetic, natural product, or organic chemical, is about the sameas for an antibody or polypeptide, on a moles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with autoimmunedisease or pathogen-induced immunopathology and/or a reduction in theseverity of such symptoms that will or are expected to develop. Theterms further include ameliorating existing uncontrolled or unwantedautoimmune-related or pathogen-induced immunopathology symptoms,preventing additional symptoms, and ameliorating or preventing theunderlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with aninflammatory disease.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an anti-TSLP antibody orfragment thereof, that when administered alone or in combination with anadditional therapeutic agent to a cell, tissue, or subject is effectiveto prevent or ameliorate the autoimmune disease or pathogen-inducedimmunopathology associated disease or condition or the progression ofthe disease. A therapeutically effective dose further refers to thatamount of the compound sufficient to result in amelioration of symptoms,e.g., treatment, healing, prevention or amelioration of the relevantmedical condition, or an increase in rate of treatment, healing,prevention or amelioration of such conditions. When applied to anindividual active ingredient administered alone, a therapeuticallyeffective dose refers to that ingredient alone. When applied to acombination, a therapeutically effective dose refers to combined amountsof the active ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously. An effectiveamount of therapeutic will decrease the symptoms typically by at least10%; usually by at least 20%; preferably at least about 30%; morepreferably at least 40%, and most preferably by at least 50%.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art, see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., PA. The pharmaceutical composition of the invention mayalso contain other immunosuppressive or immunomodulating agents. Anysuitable immunosuppressive agent can be employed, including but notlimited to anti-inflammatory agents, corticosteroids, cyclosporine,tacrolimus (i.e., FK-506), sirolimus, interferons, soluble cytokinereceptors (e.g., sTNRF and sIL-1R), agents that neutralize cytokineactivity (e.g., inflixmab, etanercept), mycophenolate mofetil,15-deoxyspergualin, thalidomide, glatiramer, azathioprine, leflunomide,cyclophosphamide, methotrexate, and the like. The pharmaceuticalcomposition can also be employed with other therapeutic modalities suchas phototherapy and radiation.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans

VII. Antibody Production

For recombinant production of the antibodies of the present invention,the nucleic acids encoding the two chains are isolated and inserted intoone or more replicable vectors for further cloning (amplification of theDNA) or for expression. DNA encoding the monoclonal antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. In one embodiment, both thelight and heavy chains of the humanized anti-TSLP antibody of thepresent invention are expressed from the same vector, e.g. a plasmid oran adenoviral vector.

Antibodies of the present invention may be produced by any method knownin the art. In one embodiment, antibodies are expressed in mammalian orinsect cells in culture, such as chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, babyhamster kidney (BHK) cells, Spodoptera frugiperda ovarian (SD) cells. Inone embodiment, antibodies secreted from CHO cells are recovered andpurified by standard chromatographic methods, such as protein A, cationexchange, anion exchange, hydrophobic interaction, and hydroxyapatitechromatography. Resulting antibodies are concentrated and stored in 20mM sodium acetate, pH 5.5.

In another embodiment, the antibodies of the present invention areproduced in yeast according to the methods described in WO2005/040395.Briefly, vectors encoding the individual light or heavy chains of anantibody of interest are introduced into different yeast haploid cells,e.g. different mating types of the yeast Pichia pastoris, which yeasthaploid cells are optionally complementary auxotrophs. The transformedhaploid yeast cells can then be mated or fused to give a diploid yeastcell capable of producing both the heavy and the light chains. Thediploid strain is then able to secret the fully assembled andbiologically active antibody. The relative expression levels of the twochains can be optimized, for example, by using vectors with differentcopy number, using transcriptional promoters of different strengths, orinducing expression from inducible promoters driving transcription ofthe genes encoding one or both chains.

In one embodiment, the respective heavy and light chains of theanti-TSLP antibody are introduced into yeast haploid cells to create alibrary of haploid yeast strains of one mating type expressing aplurality of light chains, and a library of haploid yeast strains of adifferent mating type expressing a plurality of heavy chains. Theselibraries of haploid strains can be mated (or fused as spheroplasts) toproduce a series of diploid yeast cells expressing a combinatoriallibrary of antibodies comprised of the various possible permutations oflight and heavy chains. The combinatorial library of antibodies can thenbe screened to determine whether any of the antibodies has propertiesthat are superior (e.g. higher affinity for TSLP) to those of theoriginal antibodies. See. e.g., WO2005/040395.

In another embodiment, antibodies of the present invention are humandomain antibodies in which portions of an antibody variable domain arelinked in a polypeptide of molecular weight approximately 13 kDa. See,e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain, lowmolecular weight agents provide numerous advantages in terms of ease ofsynthesis, stability, and route of administration.

VIII. Uses

The present invention provides methods for using engineered anti-TSLPfor the treatment and diagnosis of inflammatory disorders.

In a preferred embodiment, the inflammatory disorder is asthma.

In another preferred embodiment, the inflammatory disorder is anallergic inflammatory disorder. In a preferred embodiment, the allergicinflammatory disorder is allergic rhinosinusitis, allergic asthma,allergic conjunctivitis, or atopic dermatitis.

The present invention provides methods for using engineered anti-TSLPfor the treatment and diagnosis of fibrosis, inflammatory bowel disease,Hodgkin's lymphoma, respiratory viral infections or other viralinfections, rheumatoid arthritis, or any other disorder characterized byinflammation at the site of injury.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

EXAMPLE 1 General Methods

Standard methods in molecular biology are described (Maniatis et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif.). Standard methods also appear in Ausbel et al.(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, JohnWiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocolsin Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life ScienceResearch, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001)BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification,and fragmentation of polyclonal and monoclonal antibodies are described(Coligan et al. (2001) Current Protcols in Immunology, Vol. 1, JohnWiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlowand Lane, supra). Standard techniques for characterizing ligand/receptorinteractions are available (see, e.g., Coligan et al. (2001) CurrentProtcols in Immunology, Vol. 4, John Wiley, Inc., New York).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742;Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren etal. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

EXAMPLE 2 Humanization of Anti-Human TSLP Antibodies

Rat anti-human TSLP antibody 23B12 is produced by the hybridomadeposited at the American Type Culture Collection (10801 UniversityBlvd., Manassas, Va. 20110-2209 USA) (“ATCC”) with the patent depositdesignation “PTA-7951.” The hybridoma was deposited on Oct. 26, 2006under the conditions of the Budapest Treaty, and received accessionnumber PTA-7951. The humanization of rat anti-human TSLP antibody 23B12was performed as essentially as described in PCT patent applicationpublications WO 2005/047324 and WO 2005/047326, which are incorporatedby reference.

Variable light and heavy domains of the anti-TSLP monoclonal antibody(23B12) were cloned and fused to a human kappa light chain (CL domain)and human IgG1 heavy chain (CH1-hinge-CH2-CH3), respectively.

The amino acid sequence of the non-human VH domain was compared to agroup of three human VH germline amino acid sequences; onerepresentative from each of subgroups IGHV1, IGHV3 and IGHV4. The VHsubgroups are listed in M.-P. Lefranc, “Nomenclature of the HumanImmunoglobulin Heavy (IGH) Genes”, Experimental and ClinicalImmunogenetics, 18:100-116, 2001. Rat 23B12 antibody scored highestagainst human heavy chain germline DP-14 in subgroup VH1.

For the rat 23B12 antibody, the VL sequence was of the kappa subclass ofVL. The amino acid sequence of the non-human VL domain was compared to agroup of four human VL kappa germline amino acid sequences. The group offour is comprised of one representative from each of four establishedhuman VL subgroups listed in V. Barbie & M.-P. Lefranc, “The HumanImmunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ) Segments”,Experimental and Clinical Immunogenetics, 15:171-183, 1998 and M.-P.Lefranc, “Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes”,Experimental and Clinical Immunogenetics, 18:161-174, 2001. The foursubgroups also correspond to the four subgroups listed in Kabat et al.“Sequences of Proteins of Immunological Interest”, U.S. Department ofHealth and Human Services, NIH Pub. 91-3242, 5th Ed., 1991, pp. 103-130.Rat 23B12 antibody scored highest against human light chain germlineZ-A27 in subgroup VLkIII.

Once the target amino acid sequences of the variable heavy and lightchains were determined, plasmids encoding the full-length humanizedantibody were generated. Starting with a plasmid encoding a humanizedanti-IL-23 antibody having VH1 DP-14 germline framework and a separateplasmid encoding a humanized anti-IGFR antibody having VLkIII Z-A27germline framework, the plasmids were altered using Kunkel mutagenesis(see, e.g., Kunkel T A. (1985) Proc. Natl. Acad. Sci. U.S.A 82:488-492)to change the DNA sequence to the target humanized 23B12 sequence.Simultaneously, codon optimization was incorporated into the changes toprovide for potentially optimal expression. The humanized heavy andlight variable chain amino acid sequences, are set forth in SEQ ID NOs:10 and 12. The full-length humanized heavy and light chain amino acidsequences, are set forth in SEQ ID NOs: 14 and 16. A variant of thelight chain was also created, wherein the variant comprised a K (ratherthan a Y) at amino acid position 49 of SEQ ID NO:12 or SEQ ID NO:16.

EXAMPLE 3 Determining the Equilibrium Dissociation Constant (K_(D)) forHumanized Anti-Human TSLP Using KinExA Technology

The equilibrium dissociation constant (K_(D)) was determined using theKinExA 3000 instrument (Sapidyne Instruments Inc., www.sapidyne.com).The KinExA uses the principle of the Kinetic Exclusion Assay methodbased on measuring the concentration of uncomplexed antibody in amixture of antibody, antigen and antibody-antigen complex. Theconcentration of free antibody is measured by exposing the mixture to asolid-phase immobilized antigen for a very brief period of time. Inpractice, this is accomplished by flowing the solution phaseantigen-antibody mixture past antigen-coated particles trapped in a flowcell. Data generated by the instrument are analyzed using customsoftware. Equilibrium constants are calculated using a mathematicaltheory based on the following assumptions:

1. The binding follows the reversible binding equation for equilibrium:k _(on) [Ab][Ag]=k _(off) [AbAg]

2. Antibody and antigen bind 1:1 and total antibody equalsantigen-antibody complex plus free antibody.

3. Instrument signal is linearly related to free antibody concentration.

Materials

Antibodies:

Rat anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330)

Rat anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330A)

Humanized anti hu TSLP mAb 23B12 (VL Y49)

Humanized anti hu TSLP mAb 23B12 (VL K49)

Humanized anti hu TSLP mAb 23B12 (VL Y49)

Antigens:

Recombinant human TSLP (SPB Lot P345)

Recombinant human TSLP (SPB Lot P367)

Recombinant human TSLP (R&D, Cat.N. 1398-TS/CF, Lot IDK 015031)

Biotinylated Antigens:

Biotinylated human TSLP (SPB Lot p367AC)

Biotinylated human TSLP (SPB Lot p367AA)

Biotinylated human TSLP (SPB Lot 38ABMA)

Other Reagents:

PMMA particles, 98 micron (Sapidyne, Cat No. 440198)

Neutravidin (Pierce, Cat No. 31000)

Cy5-conjugated Goat anti-rat IgG (H+ L) (Jackson ImmunoresearchLaboratories Cat. No 112-175-167, Lot 60306)

Cy5-conjugated Goat anti-HuIgG (H+ L) (Jackson ImmunoresearchLaboratories Cat. No 109-175-088, lot 49069 and lot 58552)

Experimental Conditions:

PMMA particles were coated with biotinylated human TSLP according toSapidyne “Protocol for coating PMMA particles with biotinylated ligandshaving short or nonexistent linker arms”. All experimental procedureswere done according to the KinExA 3000 manual. All runs were done induplicate.

For Rat Anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330) the FollowingConditions were Used:

Sample volume: 2 mL

Sample flow rate: 0.25 mL/min

Label volume: 1 mL

Label flow rate: 0.25 mL/min

Antibody conc.: 0.1 nM

Highest antigen conc.: 10 nM

Lowest antigen conc.: 10 pM

For Rat Anti hu TSLP mAb 23B12.H8.A4 (SPB Lot PAB330A) the FollowingConditions were Used:

Sample volume: 4 mL

Sample flow rate: 0.25 mL/min

Label volume: 1 mL

Label flow rate: 0.25 mL/min

Antibody conc.: 0.05 nM

Highest antigen conc.: 0.5 nM

Lowest antigen conc.: 0.5 pM

For Humanized Anti hu TSLP mAbs the Following Conditions were Used:

Sample volume: 2 mL

Sample flow rate: 0.25 mL/min

Label volume: 1 mL

Label flow rate: 0.25 mL/min

Antibody conc.: 0.02 nM

Highest antigen conc.: 0.2 nM

Lowest antigen conc.: 0.2 pM

-   -   Two-fold serial dilutions of the antigen were prepared and mixed        with the antibody at constant concentration. The mixture was        incubated for 2 hours at 25° C. to equilibrate.

Table 4 shows the results of the KinExA analysis.

TABLE 4 K_(D) Values Determined by KinExa mAb TSLP Expression K_(D) (pM)rat 23B12.H8.H4 human HEK293 0.22 rat 23B12.H8.H4 human E. coli 0.47hu23B12(VL Y49) human HEK293 2.1 hu23B12(VL K49) human HEK293 1.0hu23B12(VL Y49) human E. coli 1.7

EXAMPLE 4 Determining the EC₅₀ for Humanized Anti-Human TSLP Using ELISA

The ELISA measures the EC50 of rat 23B12 purified from hybridomasupernatant or recombinant humanized 23B12 IgG1 binding to eitheradenovirus-derived human TSLP (S-P Biopharma) or E. coli-derived humanTSLP (S-P Biopharma or R&D 1398-TS).

Materials:

Nunc Maxisorb 96 well Immunoplate cert. (Nunc #439454)

OX phosphate-buffered saline (PBS), pH 7.4 (Fisher # BP399-20)

20× Tris Buffered Saline (TBS), pH 7.4 (Technova #1680)

Tween-20, enzyme grade (Fisher # BP337-500)

500 mM EDTA (Technova # E0306)

Albumin, bovine serum RIA grade (Sigma # A7888)

Coating Buffer: 1 μg/mL TSLP in PBS at 100 μL/well

Detection Reagent:

HRP—F(ab)′2 goat anti-human IgG H+ L (Jackson #109-036-088);

HRP—F(ab)′2 goat anti-rat IgG H+ L (Jackson #112-032-072)

Substrate & Stop Solutions:

TMB Microwell Peroxidase Substrate System 2C (Kirkegaard & Perry Labs#50-76-00) 1:1; 1M H₃PO₄ 0.1 mL/well

ABTS (Kirkegaard & Perry Labs #50-66-06) 100 μL/well

ABTS Peroxidase stop solution (Kirkegaard & Perry Labs #50-085-02) 5×concentrate diluter 1:5 in MIlli-Q water, 100 μL/well

ELISA Diluent and Assay Buffer:

50 mM TBS or PBS; 0.5% BSA; 0.05% Tween-20; 4 mM EDTA

ELISA Wash Buffer:

50 mM TBS or PBS; 0.05% Tween-20; 4 mM EDTA

Equipment:

Skatron Scanwasher300™

Molecular Devices VersaMax™ microplate reader

Protocol

Coating of plates was performed as follows: TSLP (100 or 200 ng perwell) in PBS was incubated at 4° C. overnight. Plates were washed with 1cycle (4 washes/cycle) on a Skatron plate washer, blocked by addition of0.2 mL/well ELISA assay buffer, incubated for 60 min at 25° C. on anorbital shaker and then washed for 1 cycle. Antibody was then titratedacross a row of eight wells in the range of 1000 ng/mL to 0.4572 ng/mLusing serial 3-fold dilutions and incubated for 90 min. at 25° C. on anorbital shaker. Plates were washed for 1 cycle, HRP-goat F(ab′)₂anti-human or anti-rat IgG (H+ L) (1:5,000 dilution) was added at 0.1mL/well and incubated for 60 min at 25° C. on an orbital shaker. Plateswere washed for 2 cycles with plate rotation between cycles. TMB or ABTSsubstrate was added at 0.1 mL/well and incubated 5 min on orbitalshaker. Stop solution was then added at 0.1 mL/well and the plates readat A_(450-570 nm) (TMB) or A_(405 nm)(ABTS).

Table 5 shows the results of the ELISA analysis.

TABLE 5 EC₅₀ Values Determined By ELISA TSLP TSLP mAb SpeciesExpression¹ EC₅₀ (nM) rat 23B12.H8.H4 human HEK293-B 0.79 rat23B12.H8.H4 human HEK293 0.37 rat 23B12.H8.H4 human HEK293-F 0.26 0.47 ±0.28(n = 3) rat 23B12.H8.H4 human E. coli-B 0.40 rat 23B12.H8.H4 humanE. coli-B 0.39 0.44 ± 0.20(n = 5) hu23B12(VL Y49) human HEK293-B 0.22hu23B12(VL Y49) human HEK293 0.029 hu23B12(VL Y49) human HEK293-F 0.018hu23B12(VL Y49) human HEK293 0.17 hu23B12(VL Y49) human HEK293 0.21hu23B12(VL Y49) human HEK293 0.11 hu23B12(VL Y49) human HEK293 0.13hu23B12(VL Y49) human HEK293 0.15 hu23B12(VL Y49) human HEK293 0.24hu23B12(VL Y49) human HEK293 0.18 hu23B12(VL Y49) human HEK293 0.11hu23B12(VL Y49) human HEK293² 0.073 0.14 ± 0.07(n = 12) hu23B12(VL Y49)human E. coli-B 0.064 hu23B12(VL Y49) human E. coli-B 0.085 hu23B12(VLY49) human E. coli 0.11 hu23B12(VL Y49) human E. coli 0.11 hu23B12(VLY49) human E. coli 0.060 hu23B12(VL Y49) human E. coli ² 0.061hu23B12(VL Y49) human E. coli ² 0.029 0.07 ± 0.03(n = 7) 0.11 ± 0.07(n =19) hu23B12(VL Y49) cyno HEK293 0.45 hu23B12(VL Y49) cyno HEK293 0.52hu23B12(VL Y49) cyno HEK293 0.29 hu23B12(VL Y49) cyno HEK293 0.15hu23B12(VL Y49) cyno HEK293 0.19 hu23B12(VL Y49) cyno HEK293 0.27 0.31 ±0.15(n = 6) hu23B12(VL K49) human HEK293-B 0.12 hu23B12(VL K49) human E.coli-B 0.038 hu23B12(VL K49) human E. coli-B 0.050 hu23B12(VL K49) humanHEK293 0.034 hu23B12(VL K49) human HEK293-F 0.021 0.05 ± 0.04(n = 5) ¹-B= TSLP biotinylated -F = TSLP removal of furin cleavage site viaK101A/R102A ²direct coat of 200 ng TSLP instead of 100 ng

EXAMPLE 5 Affinity of Rat 23B12 and Humanized 23B12 Antibodies for Humanand Cyno TSLP

The kinetic binding activities of the parental rat and its humanizedderivative anti human TSLP antibody 23B12 against both human (hu) andcynomolgus monkey (cyno) TSLP were measured by surface plasmon resonanceusing a BIAcore T100 system (BIAcore AB, Upsalla, Sweden). Approximately100 RUs of human TSLP or cyno TSLP were immobilized via amine couplingchemistry onto a Sensor Chip CM5 (Research grade, BR-1006-68). HBS-EPbuffer (BR-1006-69) was used as the running buffer with a flow rate of304/min. rat and humanized 23B12 antibodies at varying concentrationsranging from 0.82 to 600 nM were injected over the immobilized hu orcyno TSLP surfaces at a flow rate of 304/min. Following each injectioncycle the CM5 chip surface was regenerated using a series of solutions(10 mM Glycine pH 1.5 and 25 mM NaOH respectively) at a flow rate of754/min

Background subtraction binding sensorgrams were used for analyzing therate constant of association (ka) and dissociation (kd), and theequilibrium dissociation constant K_(D). The resulting data sets werefitted with a bivalent analyte model using the BIAevaluation software(version 1.0). The K_(D) determined for the parental rat 23B12 antibodyagainst human TSLP was 64 pM, while the respective value against thecyno TSLP ligand was 86 pM (Table 6). The K_(D) determined for thehumanized 23B12 antibody against human TSLP was 111 pM, while therespective value against the cyno TSLP ligand was 132 pM (Table 6),indicating a less than two fold loss of affinity upon humanization of23B12 mAb.

TABLE 6 BIAcore Analysis Antibody Ligand ka (1/Ms) kd (1/s) K_(D) (pM)rat 23B12 huTSLP 3.18E+05 2.1E−05 64 cynoTSLP 1.86E+05 1.6E−05 86 hu23B12 huTSLP 5.00E+05 5.6E−05 111 cynoTSLP 3.57E+05 4.7E−05 132

EXAMPLE 6 Proliferation Bioassay For The Assessment of NeutralizingAnti-TSLP Antibody

The ability of a monoclonal antibody to biologically neutralize TSLP wasassessed by the application of short-term proliferation bioassays thatutilize cells which express recombinant TSLP receptors. The transfectantBa/F3-TSLPR-IL7Ra cells proliferate in response to TSLP and the responsecan be inhibited by a neutralizing anti-TSLP antibody. Each antibody wastitrated against a concentration of TSLP chosen within the linear regionof the TSLP dose-response curve, near plateau and above the TSLP EC₅₀.Proliferation, or lack thereof, is measured by colorimetric means usingAlamar Blue, a growth indicator dye based on detection of metabolicactivity. The ability of an antibody to neutralize TSLP is assessed byits EC50 value, or concentration of antibody that induces half-maximalinhibition of TSLP proliferation.

Ba/F3 transfectants are maintained in RPMI-1640 medium, 10% fetal calfserum, 50 μM 2-mercaptoethanol, 2 mM L-Glutamine, 50 μg/mLpenicillin-streptomycin, and 10 ng/mL mouse IL-3.

Ba/F3 proliferation bioassays are performed in RPMI-1640 medium, 10%fetal calf serum, 50 pM 2-mercaptoethanol, 2 mM L-Glutamine, and 50μg/mL penicillin-streptomycin.

The assay is performed in 96-well flat bottom plates (Falcon 3072 orsimilar). All preparations of reagents and cell suspensions utilize theappropriate bioassay medium. The assay volume is 150 μL per well.Titrations of an anti-TSLP antibody are pre-incubated with TSLP for30-60 minutes at room temperature, during which time cells are prepared.Cells are added to plates following the antibody-cytokinepre-incubation. Bioassay plates are incubated in a humidified tissueculture chamber (37 C, 5% CO₂) for 40-48 hours. At the end of theculture time, Alamar Blue (Biosource Cat #DAL1100) is added and allowedto develop for 8-12 hours. Absorbance is then read at 570 nm and 600 nm(VERSAmax Microplate Reader, Molecular Probes), and an OD₅₇₀₋₆₀₀ isobtained. Duplicates or triplicates are recommended.

Cells are used in a healthy growth state, generally at densities of3-8×10⁵/mL. Cells are counted, pelleted, washed twice in bioassaymedium, and suspended to the appropriate density for plating.

TSLP was prepared to working concentration and added to first well at 75pt. Serial dilutions of 1:3 were made by titrating 25:50 μL in bioassaymedium across wells, leaving 50 μL/well. Cells were suspended to theappropriate density for plating at 100 μL per well.

The antibody was prepared to working concentration and added to thefirst well at 75 pt. Serial dilutions of 1:3 were made by titrating25:50 μL in bioassay medium across wells, leaving 50 μL per well. TSLPat the appropriate concentration was added at 50 μL per well to thewells containing the titrated antibody. Cells were suspended to theappropriate density for plating at 50 μL per well, and added followingthe antibody-cytokine pre-incubation.

Using GraphPad Prism 3.0 software, absorbance was plotted againstcytokine or antibody concentration and EC50 values were determined usingnon-linear regression (curve fit) of sigmoidal dose-response.

The assay results are shown in Table 7.

TABLE 7 Inhibition Of Proliferation TSLP TSLP mAb Species ExpressionEC50 (nM) rat 23B12.H8.H4 human HEK293 0.093 rat 23B12.H8.H4 humanHEK293 0.085 rat 23B12.H8.H4 human HEK293 0.23 rat 23B12.H8.H4 humanHEK293 0.040 rat 23B12.H8.H4 human HEK293 0.10 0.11 ± 0.07(n = 5) rat23B12.H8.H4 human E. coli 2.16 rat 23B12.H8.H4 human E. coli 2.78 rat23B12.H8.H4 human E. coli 4.15 rat 23B12.H8.H4 human E. coli 3.81 rat23B12.H8.H4 human E. coli 1.83 rat 23B12.H8.H4 human E. coli 3.46 rat23B12.H8.H4 human E. coli 2.77 rat 23B12.H8.H4 human E. coli 3.10 3.01 ±0.79(n = 8) rat 23B12.H8.H4 cyno HEK293 0.45 rat 23B12.H8.H4 cyno HEK2930.42 rat 23B12.H8.H4 cyno HEK293 0.61 rat 23B12.H8.H4 cyno HEK293 0.770.56 ± 0.16(n = 4)

EXAMPLE 7 Neutralizing Activity of Anti-TSLP mAb r23B12 On TSLP InducedTARC Production by Human Primary Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from buffycoats obtained from healthy blood donors (Stanford Medical School BloodCenter, Stanford, Calif.) by Ficoll centrifugation and CD11c+ DendriticCells were obtained by MACS (Miltenyi Biotech, Auburn, Calif.) usingnegative selection followed by cell sorting using a FACS. Lineagenegative (Lin⁻) cells were obtained by MACS depletion of T cells, Bcells, NK cells, red blood cells and monocytes form PBMC using mouseanti-human CD3 mAb (OKT3, DNAX) and mouse anti-CD16 mAb and goatanti-mouse IgG coated magnetic beads (Miltenyi Biotech), and usingmagnetic beads directly coated with anti-CD19, CD56 and CD14 mAbs(Miltenyi Biotech). Subsequently, Lin⁻ cells were stained withTC-anti-CD4 (Caltag, Burlingame, Calif.), PE-anti-CD11c andFITC-anti-CD3, -CD14, -CD19, -CD56, -CD16, and -CD20 (all BDBiosciences, San Diego, Calif.) and CD11c+ DC sorted on a VantageFACsorter™ (BD Biosciences) to a purity>99% of CD11c⁺ CD4⁺ Lin⁻ cells.

CD11c⁺ CD4⁺ DCs were cultured immediately after sorting in RPMI(Mediatech, Herndon, Va.) containing 10% FCS and 1% pyruvate(Mediatech), HEPES (Invitrogen, Grand Island, N.Y.) andpenicillin-streptomycin (Mediatech). Cells were seeded at 0.5×10⁶/ml inflat-bottomed 96-well plates in the presence of medium alone, TSLP (15ng/ml, DNAX), or in a combination of TSLP and the neutralizing anti-TSLPmAb (clone 23B12) or an anti-TSLPR monoclonal antibody or an isotypecontrol rat IgG2a (R&D Systems, Minneapolis, Minn.). DC culturesupernatants were collected after 24 h of culture, stored frozen at −20°C. and analyzed for TARC protein levels by ELISA (R&D Systems).

The results are provided in Table 8.

TABLE 8 TARC (pg/ml) DC 5 TSLP-DC 1400.5 TSLP + 5 μg/ml r23B12 antibody41.5 TSLP + 0.5 μg/ml r23B12 antibody 146 TSLP + 0.05 μg/ml r23B12antibody 570.5 TSLP + 20 μg/ml r23B12 antibody 199

CD11c+ DC cultured in media alone do not produce significant levels ofTARC. The addition of TSLP (15 ng/ml) to CD11c+ DC induced significantlevels of TARC production up to ˜1500 pg/ml. This TSLP mediatedinduction of TARC was blocked in a dose dependent manner by thesimultaneous addition of anti-TSLP mAb 23B12.

EXAMPLE 8 Neutralizing Activity of Anti-TSLP mAb r23B12 On TSLP InducedTh2 Differentiation by Human Primary Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from buffycoats obtained from healthy blood donors (Stanford Medical School BloodCenter, Stanford, Calif.) by Ficoll centrifugation and CD11c+DendriticCells were obtained by MACS (Miltenyi Biotech, Auburn, Calif.) usingnegative selection followed by cell sorting using a FACS. Lineagenegative (Lin⁻) cells were obtained by MACS depletion of T cells, Bcells, NK cells, red blood cells and monocytes form PBMC using mouseanti-human CD3 mAb (OKT3, DNAX) and mouse anti-CD16 mAb and goatanti-mouse IgG coated magnetic beads (Miltenyi Biotech), and usingmagnetic beads directly coated with anti-CD19, CD56 and CD14 mAbs(Miltenyi Biotech). Subsequently, Lin⁻ cells were stained withTC-anti-CD4 (Caltag, Burlingame, Calif.), PE-anti-CD11c andFITC-anti-CD3, -CD14, -CD19, -CD56, -CD16, and -CD20 (all BDBiosciences, San Diego, Calif.) and CD11c+ DC sorted on a VantageFACsorter™ (BD Biosciences) to a purity>99% of CD11c⁺ CD4⁺ Lin⁻ cells.

CD11c⁺ CD4⁺ DCs were cultured immediately after sorting in RPMI(Mediatech, Herndon, Va.) containing 10% FCS and 1% pyruvate(Mediatech), HEPES (Invitrogen, Grand Island, N.Y.) andpenicillin-streptomycin (Mediatech). Cells were seeded at 0.5×10⁶/ml inflat-bottomed 96-well plates in the presence of medium alone, TSLP (15ng/ml, DNAX), or in a combination of TSLP and the neutralizing anti-TSLPmAb (clone 23B12) or the anti-TSLPR monoclonal antibody or an isotypecontrol rat IgG2a (R&D Systems, Minneapolis, Minn.). CD11c⁺ DCs werecollected after 24 h of culture under the different conditions, washedtwice and recultured with allogeneic CD4⁺ CD45RA⁺ naïve T cells.

CD4⁺ CD45RA⁺ naïve T cells were isolated by cell sorting after negativedepletion of CD8, CD16, CD20, CD19, CD56 and CD14 cells using magneticbeads (Myltenyi Biotech). After 24 h of culture under differentconditions, CD11c⁺ DCs were collected, washed twice, and co-culturedwith 5×10⁴ allogeneic naive CD4⁺ T cells in round-bottomed 96-wellplates at a ratio of 1:5 DC:T cells. After 6 d. of culture, supernatantswere collected and frozen at −20° C. and numbers of viable cellsdetermined by trypan blue exclusion. To test their capacity to secretecytokines, DC-primed CD4⁺ T cells (10⁶/ml) were restimulated withbiotinylated anti-CD3 (10 ng/ml) mAbs crosslinked to streptavidin coatedtissue culture plates in the presence of soluble anti-CD28 mAbs (1000ng/ml). Culture supernatants were collected after 24 hrs of culture,frozen at −20° C. or analyzed by Luminex assay for IFNγ-, TNFα-, IL-2,IL-4, IL-5, IL-10 and IL-13 (Linco Research Inc., St Charles, Mo.).

The results are provided in Table 9.

TABLE 9 TSLP-DC + TSLP-DC + TSLP-DC + TSLP- 5 μg/ml 0.5 μg/ml 0.05 μg/mlCytokine DC + DC + r23B12 + r23B12 + r23B12 + Production CD4 CD4 CD4 CD4CD4 IL-5 (pg/ml) 115 777 24 70.6 32 IL-4 (pg/ml) 14 89 14.5 21.3 27.3IL-13 136 1290 55.5 182 43.3 (pg/ml)

The coculture of naïve CD4+ CD45RA+ T cells with CD11c+ DC that had beencultured in media alone resulted in a T cell population, that uponreactivation by anti-CD3+anti-CD28 mAbs, produced low levels of Th2cytokines. Addition of TSLP (15 ng/ml) to the primary CD11c+ DC cultureinduced the production of significant levels of IL-4, IL-5 and IL-13 bythe responding T cells, suggesting that TSLP-DC induced thedifferentiation of naïve T cells towards Th2 cells. This TSLP mediatedinduction of Th2 differentiation was blocked in a dose dependent mannerby the simultaneous addition of anti-TSLP mAb 23B12 to the primary DCcultures.

EXAMPLE 9 Cyno-ization of Anti-Human TSLP Antibodies

Two studies have shown that cynomolgus monkeys (Macaca fascicularis) VLare similar to human VLκ-I and that cynomolgus VH are similar to humanVH-III (41%), VH-IV (39%), and VH-I (14%). (Lewis et al., Dev. Comp.Immunol. 17:549-560 (1993); and Druar et al., Immunogentics 57:730-738(2005.) In order to minimize potential immunogenicity of hu23B12 incymomolgus monkeys, the rat 23B12 CDRs were transferred onto human VLκ-Iand VH-III frameworks; these were then fused onto cynomolgus IgGconstant domains.

The amino acid sequence of the cyno-ized light chain isMAPVQLLGLLVLFLPAMRCDIQMTQSPSSLSASVGDRVTITCRASQPISISVHWYQQKPGKAPKLLIYFASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTFSLPYTFGQGTKVEIKRTVAAPSVFIFPPSEDQVKSGTVSVVCLLNNFYPREASVKWKVDGVLKTGNSQESVTEQDSKDNTYSLSSTLTLSSTDYQSHNVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17).The signal sequence is underlined.

The amino acid sequence of the cyno-ized heavy chain isMAVLGLLFCLVTFPSCVLSQVQLVESGGGVVQPGRSLRLSCAASGYIFTDYAMHWVRQAPGKGLEWVATFIPLLDTSDYNQNFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGVTHSYVMDAWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGSLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYVCNVNHKPSNTKVDKRVEIKTCGGGSKPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHHAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKALPAPIQKTISKDKGQPREPQVYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQID NO:18). The signal sequence is underlined.

Cyno-ized anti-human TSLP 23B12 antibodies were then recombinantlyproduced in CHO cells.

EXAMPLE 10 Proliferation Bioassay for the Assessment of the NeutralizingActivity of Cyno-ized Anti-Human TSLP Antibodies

The ability of cyno-ized anti-human TSLP 23B12 antibodies tobiologically neutralize human or cyno TSLP was assessed by theapplication of short-term proliferation bioassays that utilize cellswhich express recombinant TSLP receptors. The transfectantBa/F3-huTSLPR/huIL-7Ra and Ba/F3-cyTSLPR/cyIL-7Ra cells proliferate inresponse to human TSLP and cyno TSLP, and the response can be inhibitedby a neutralizing anti-TSLP antibody. An antibody is titrated against aconcentration of human or cyno TSLP chosen within the linear region ofthe dose-response curve, near plateau and above EC50. Proliferation, orlack thereof, is measured by colorimetric means using Alamar Blue, agrowth indicator dye based on detection of metabolic activity. Theability of an antibody to neutralize TSLP is assessed by its EC50 value,or concentration of antibody that induces half-maximal inhibition ofTSLP proliferation.

Ba/F3 transfectants are maintained in RPMI-1640 medium, 10% fetal bovineserum, 50 uM 2-mercaptoethanol, 2 mM L-Glutamine, 50 ug/mLpenicillin-streptomycin, 10 ng/mL mouse IL-3, 1 mg/ml G418, and 2 ug/mlpuromycin.

Ba/F3 proliferation bioassays are performed in RPMI-1640 medium, 10%fetal bovine serum, 50 uM 2-mercaptoethanol, 2 mM L-Glutamine, and 50ug/mL penicillin-streptomycin.

The assays were performed in 96-well flat bottom plates (Falcon 3072 orsimilar). All preparations of reagents and cell suspensions utilized theappropriate bioassay medium. The assay volume was 150 uL per well.Titrations of an anti-TSLP antibody were pre-incubated with huTSLP orcyTSLP for approximately 30 minutes at room temperature, during whichtime cells were prepared. Cells were added to plates following theantibody-cytokine pre-incubation. Bioassay plates were incubated in ahumidified tissue culture chamber (37 C, 5% CO₂) for 40-48 hr. At theend of the culture time, Alamar Blue (Biosource Cat #DAL1100) was addedat 16.5 uL/well and allowed to develop for 5-12 hours. Absorbance wasthen read at 570 nm and 600 nm (VERSAmax Microplate Reader, MolecularDevices), and an OD₅₇₀₋₆₀₀ was obtained. Duplicates were run for eachsample.

Cells are used in a healthy growth state, generally at densities of7−9×10⁵/mL. Cells are washed twice in bioassay medium, counted, andsuspended to the appropriate density for plating at 7500 cells/50 ul perwell.

Human or cyno TSLP was prepared to working concentration (600 ng/mL) andadded to first well at 75 uL. Serial dilutions of 1:3 were made bytitrating 25:50 uL in bioassay medium across wells, leaving 50 uL/well.Cells were suspended to the appropriate density for plating at 7500cells/50 uL per well. To substitute the addition of antibody, 50 ul ofbioassay medium was added to these wells to bring the final volume to150 ul.

The antibody was prepared to working concentration (3× the finalconcentration; the final starting concentration of each antiboedyvaried) and added to first well at 75 uL. Serial dilutions of 1:3 weremade by titrating 25:50 uL in bioassay medium across wells, leaving 50uL per well. TSLP (at working concentration of 9 ng/ml for HuTSLP, 3ng/ml for CyTSLP) was added at 50 uL per well to the wells containingthe titrated antibody. Cells were then suspended to the appropriatedensity for plating at 7500 cells/50 uL per well, and added followingthe antibody-cytokine pre-incubation.

EC50 values are determined by non-linear regression (curve fit) ofsigmoidal dose-response using GraphPad Prism 4 software. For TSLP doseresponse, absorbance is plotted against cytokine concentration. Forneutralization activity, percentage inhibition is plotted againstantibody concentration.

The assay results are shown in Table 10.

TABLE 10 Ba/F3 Cell-based Assay of Human or Cynomolgus TSLPR-TransfectedCells EC50 nM TSLP TSLPR rat23B12 hu23B12 hu-cy* cy-hu** cy23B12 hu hu0.6 7.6 5.3 3.9 9.4 hu hu 1.2 9.0 hu hu 0.1 1.2 hu cyno 0.02 0.03 hucyno 0.08 0.05 cyno cyno 0.2 0.5 0.5 2.8 4.7 cyno cyno 0.4 2.2 3.2 1626, 19 cyno cyno 0.7 3.0 22, 17 *hu-cy = humanized 23B12 VL/VH oncynomolgus constant domains **cy-hu = cyno-ized 23B12 VL/VH on humanconstant domains

EXAMPLE 11 BIAcore and KinExA Affinity Measurements for Cyno-izedAnti-TSLP Antibodies

The affinity of cyno-ized anti-human TSLP 23B12 antibodies towards humanand cyno TSLP ligand was determined by surface plasmon resonance usingthe BIAcore

T100 system as described in Example 5.

The equilibrium disassociation constant for the anti-TSLP antibodies wasdetermined using the KinExA 3000 instrument (Sapidyne Instruments Inc.).as described in Example 3.

The Following Materials were Used:

Antibodies:

Rat anti hTSLP GNE01.23B12.H8.A4 (SPB Lot pab 330A)

Humanized anti hTSLP mAb 23B12(621HC/780LC)

Cynoized anti hTSLP mAb 23B12 (782+MAFA19/781MAFA7)

Cynoized anti hTSLP mAb 23B12 (782+hIgG1/781hukappa)

Cynoized anti hTSLP mAb 23B12 (huV-CynoC chimera)

Antigens:

Recombinant human TSLP, R&D Systems (Cat. No. 1398-TS/CF, Lot. IDK015031)

Recombinant human TSLP, R&D Systems (Cat. No. 1398-TS, Lot. IDK 026031)

Biotinylated human TSLP (SPB Lot 38ABMA)

Other Reagents:

PMMA particles, 98 micron (Sapidyne, Cat No. 440198)

Neutravidin (Pierce, Cat No. 31000)

Cy5 conjugated Goat anti-rat IgG (H+ L) (Jackson ImmunoresearchLaboratories Cat. No 112-175-167, Lot 60306)

Cy5 conjugated Goat anti-huIgG (H+ L) (Jackson ImmunoresearchLaboratories Cat. No 109-175-088, lot 58552)

For r23B12 the Following Conditions were Used:

Sample volume: 2 ml

Sample flow rate: 0.25 ml/min

Label volume: 1 ml

Label flow rate: 0.25 ml/min

mAb conc.: 0.05 nM

Highest Ag (TSLP) conc.: 0.5 nM

Lowest Ag (TSLP) conc.: 0.5 pM

For hu23B12 the Following Conditions were Used:

Sample volume: 2 ml

Sample flow rate: 0.25 ml/min

Label volume: 1 ml

Label flow rate: 0.25 ml/min

mAb conc.: 0.02 nM

Highest Ag (TSLP) conc.: 0.4 nM

Lowest Ag (TSLP) conc.: 0.4 pM

For cy23B12 the Following Conditions were Used:

Sample volume: 2 ml

Sample flow rate: 0.25 ml/min

Label volume: 1 ml

Label flow rate: 0.25 ml/min

mAb conc.: 0.1 nM

Highest Ag (TSLP) conc.: 1 nM

Lowest Ag (TSLP) conc.: 1 pM

For hu-cy23B12* and cy-hu23B12** the following conditions were used:

Sample volume: 2 ml

Sample flow rate: 0.25 ml/min

Label volume: 1 ml

Label flow rate: 0.25 ml/min

mAb conc.: 0.05 nM

Highest Ag (TSLP) conc.: 1 nM

Lowest Ag (TSLP) conc.: 1 pM

For all experiments two-fold serial dilutions of the antigen wereprepared and mixed with the antibody at constant concentration. Themixture was incubated for 2 hours at RT to equilibrate. The results ofthe BIAcore and KinExA experiments described above are summarized onTable 11.

TABLE 11 Biacore and Kinexa Binding Affinity Measurements KD (pM) TSLPr23B12 hu23B12 hu-cy* cy-hu** cy23B12 KinExA hu 0.47 1.7 1.4 63 52BIAacore hu 64 111 106 556 620 hu 126, 114 1548, 2066 cy  86, 112 132114 1203 1159, 2508 *hu-cy = humanized 23B12 VL/VH on cynomolgusconstant domains **cy-hu = cynoized 23B12 VL/VH on human constantdomains

In summary, the humanized anti-human TSLP 23B12 antibody showedapproximately 5-fold reduced binding compared to the parental ratantibody based on BIAcore and KinExA measurements (Table 11). Replacingthe humanized 23B12 frameworks (VLκ-III/VH-I) with those lesspotentially immunogenic in cynomolgus monkeys (VLκ-I/VH-III) effected a10-fold reduction in binding compared to parental rat 23B12 and a 5-foldreduction compared to hu23B12 (Tables 10, 11).

EXAMPLE 12 Pharmacokinetic Studies of Cyno-ized Anti-TSLP 23B12Antibodies

An ELISA assay was designed to measure the amount of cyno-ized anti-TSLPantibody reaching the plasma, serum or bronchoalveolar lavage (BAL)fluid of an animal inoculated with such an antibody.

Reagents and Buffers:

-   -   Solid Support: Nunc Maxisorp 96-Well plate (cat#439454)    -   Coating Buffer: 50 mM Sodium carbonate/bicarbonate pH9.6    -   Blocking Buffer: 0.5% BSA in PBS    -   Assay Diluent Buffer 0.5% BSA [wt/v], 0.05% Tween 20 [v/v],        0.25% CHAPS [wt/v], 5 mM EDTA, 0.35M NaCl in PBS (AD), pH7.4    -   Wash Buffer: 0.05% Tween 20 in PBS    -   Capturing molecule: huTSLP, 38ABM, 2497 μg/mL    -   Detection molecules:        -   QED R799, 3600 ug/mL (a rabbit polyclonal anti-cy23B12            antibody)        -   anti rabbit-HRP, JIR cat#711-036-152    -   Substrate: TMB (Kirkegaard & Perry, cat#50-76-03)    -   Stop solution: 1M H3PO4    -   Plate Washer SkanWasher 300 Model 12010 (Molecular Devices Cat.        No. 0200-3903)    -   Stop solution: SpectraMax Plus 384 Microtiterplate        Spectrophotometer (Molecular Devices Part No. 0112-0056    -   Protocol:

Coating of plates was performed as follows: huTSLP (100 ng per well) incoating buffer was incubated at 40 C overnight. Plates were washed with1 cycle (3 washes/cycle) on a Skatron plate washer, blocked by additionof 150 μL/well blocking buffer, incubated for 60 min at room temperatureon an orbital shaker and then washed for 1 cycle. The cyno-izedanti-TSLP 23B12 antibody standard was titrated across a row of eightwells (replicates) in the range of 200 ng/mL to 1.56 ng/mL using serial2-fold dilutions. Samples are serially diluted respect to their expectedlevels. 100 μL of standards, controls, and samples were added to thecoated plate and incubated for 120 minutes at room temperature on anorbital shaker. Plates were washed for 2 cycles and the rabbitpolyclonal anti-cy23B12 antibody was added at 100 μL/well and incubatedfor 60 min at room temperature on an orbital shaker. Plates were washedfor 2 cycles, HRP-donkey anti-rabbit IgG (H+ L) (1:10,000 dilution) wasadded at 100 μL/well and incubated for 60 min at room temperature on anorbital shaker. Plates were washed for 2 cycles with plate rotationbetween cycles. TMB substrate was added at 100 μL/well and incubatedapproximately 5 min on an orbital shaker. Stop solution was then addedat 100 μL/well and the plates read at A450-650 nm (TMB).

This assay can detect as low as 156 ng/mL of cyno-ized anti-TSLPantibodies in plasma (5% dilution) and serum; and as low as 3.2 ng/mL inBAL fluid.

This ELISA assay was used to measure the pharmacokinetics of cyno-izedanti-TSLP 23B12 antibodies after administration to mice and monkeys.

A single dose PK study was conducted in normal CD-1 mice. In this study,ten mice received 10 mg/kg of the antibody by intravenous (IV)administration; and ten mice received 10 mg/kg of the antibody bysubcutaneous (SC) administration. The results of the study aresummarized in Table 12.

TABLE 12 AUC Clearance Vss 0-last t½ (mL/ (mL/ (μg*day/ terminal TmaxCmax F Route day/kg) kg) mL) (day) (day) (μg/mL) (%) IV 24.8 220 3666.69 — — — IV (w/o 22.8 198 404 6.36 Day 10) SC — — 370 3.13 0.667 73.795.6

Table 13 summarizes the percentage of cyno-ized anti-TSLP antibody foundin the BAL fluid versus in the serum at various time points afeter IV orSC administration.

TABLE 13 IV SC Time point (day) BAL/Serum Mean (%) BAL/Serum Mean (%)0.250 1.686 4.080 1.000 2.811 5.880 3.000 5.677 7.301 7.000 13.74016.945 10.000 14.319 5.064 14.000 6.517 16.912

Two single dose PK studies were also conducted in cynomolgus monkeys.The two studies used different formulations of the cyno-ized anti-TSLP23B12 antibody: one containing 0.05% of Triton X-100 and one withoutTriton X-100. Each study contained three monkeys. The dose used in eachstudy is indicated in Table 14. The antibody was administeredsubcutaneously. The results of the studies are summarized in Tables 14and 15.

TABLE 14 AUC Ani- Dose CL/F 0-last t½ Cmax Formu- mal (mg/ (mL/ (mg*day/terminal Tmax (mg/ lation ID kg) day/kg) mL) (day) (day) mL) no Cyno 5.615.1 326 (600) 9.18 1 26.4 Triton M2-04 Cyno 5.6 7.96  594 (1090) 10 148.3 M7-05 Cyno 5.7 8.82 491 (887) 12.4 2 34 M21-05 0.05% Cyno 10.7 10.3666 8.15 4 61.6 Triton 2172 X100 Cyno 10.3 8.27 778 8.86 2 88.8 3048Cyno 9.8 8.14 753 9.08 1 89.9 5102

TABLE 15 Formulation BAL/Serum Ratio (%) Time point (day) M2-04 M7-05M21-05 Mean No Triton  3 0.657 0.712 0.335 0.568 10 1.83  1.15  1.81 1.60  Time point (day) 2172 3048 5102 Mean +0.05%  3 0.313 0.492 1.39 0.733 Triton X100 10 0.090 0.620 0.693 0.467

EXAMPLE 13 Administration of Cyno-ized anti-TSLP 23B12 Antibodies toCynomolgus Monkeys

Cyno-ized anti-TSLP 23B12 antibodies were produced from a stablytransfected CHO cell line in suspension culture. The supernantant washarvested, contreated, and purified using several standardchromatogrphic steps to achieve a low endotoxin, >95% pure preparation.The purified antibody was formulated for stability during handling anduse, including multiple freeze-thaws, in 20 mM sodium acetate, pH 5.5,7% (w/v) sucrose, and 0.05% Triton X-100. This antibody is to beadministered to house dust mite (HDM) allergic cynomolgus monkeys todemonstrate the effectiveness of an anti-TSLP antibodies to treatallergic lung inflammation. This animal model will make possible thecollection of airway tissues, BAL fluid, and associated PBMC's harvestedfrom the control and cy23B12 treated animals; and will provide theability to access efficacy in Early Allergic Reactions (EAR) and LateAllergic Reactions (LAR). Further information regarding non-primatemodels of chronic allergic asthma are well known in the art. See, e.g.,Schelegle et al., Am. J. Pathology 158(1):333-341 (2001); Avdalovic etal., Am. J. Respir. Crit. Care Med. 174:1069-74 (2006) Care and VanScott et al., J. Appl. Physiol. 99(6):2080-2086 (2005).

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. U.S. patents and other publicationsreferenced herein are hereby incorporated by reference.

What is claimed is:
 1. A method of suppressing a Th2-mediated immuneresponse in a human subject comprising administering to a subject inneed thereof an isolated antibody that specifically binds human and cynoTSLP, or a TSLP-binding fragment thereof, in an amount effective toblock the biological activity of TSLP; wherein the antibody is selectedfrom the group consisting of: (i) an antibody comprising the three CDRsequences set forth in SEQ ID NOs: 1, 2 and 3 and the three CDRsequences set forth in SEQ ID NOs: 4, 5 and 6; (ii) a monoclonalantibody that specifically binds to the epitope on human TSLP that isbound by the antibody produced by the hybridoma deposited as PTA-7951;and (iii) a monoclonal antibody that competitively inhibits binding bythe antibody produced by the hybridoma deposited as PTA-7951 to humanTSLP.
 2. The method of claim 1, wherein the immune response is anallergic inflammatory disorder.
 3. The method of claim 2, wherein thesubject has atopic dermatitis.
 4. The method of claim 2, wherein thesubject has a disorder selected from the group consisting of allergicrhinosinusitis, allergic asthma, allergic conjunctivitis, or asthma.